Indian Journal of Biotechnology
Vol 5, October 2006, pp 543-550
In vitro cloning of apple (Malus domestica Borkh) employing forced shoot tip
cultures of M9 rootstock
M Amin Dalal*, B Das, A K Sharma, M Amin Mir and Amarjeet Singh Sounduri
Biotechnology Laboratory, Division of Pomology, Sher-e-Kashmir University of Agricultural Sciences and Technology
Shalimar Campus, Srinagar 191 121, India
Received 20 February 2005; revised 27 July 2005; accepted 20 November 2005
For micropropagation of adult apple (Malus domestica Borkh) rootstock M9, explants were harvested from pre-chilled
(4±3°C) dormant cuttings forced in growth chamber. Primary explants were established by using a slightly modified version
of culture initiation procedure developed earlier. Multiple shoots, raised by axillary branching of surviving explants, were
obtained from established cultures in two cultural pathways: (i) repeat transfer of primary explants on the MS supplemented
with BAP (2.22 µM), IBA (0.49 µM) and Kn (2.2 µM), and (ii) repeat subculture of harvested microshoots and basal
portion of sectored proliferating clumps on the same but suitably PGR amended medium, in which cytokinins concentration
was reduced by half. A 4-fold shoot multiplication was achieved during each sub culture of 3±1 week’s duration that
repeatedly produced crop of proliferated shoots without loss of vigour. Nodal segments (5-15 mm), obtained from in vitro
raised microshoots were also used to initiate a new cycle of proliferating cultures. Isolated cloned microshoots (15-20 mm)
with apical bud were cultured on MS basal medium supplemented with IBA (14.70 µM) for 8±3 d to initiate root. The
microshoots were re-implanted in PGR free half strength basal MS medium with full complement of organics for
4±1 week for root development. The transferred in vitro hardened plantlets to polyvinyl cups or polybags, under carefully
controlled descending RH regime of 95% to 70±5% over a period of 5±1 week, resulted in 80% ex vitro survival. The
present protocol highlighted a novel strategy of micropropagation of apple rootstock M9 using three-step culture initiation
procedure of forced primary explants.
Keywords: apple, dwarfing rootstocks, forced explants, hardening, in vitro propagation
IPC Code: Int. Cl.8 A01H4/00
Introduction
Most commercially cultivated fruit trees have dual
genetic system, resulting from the combination of
rootstock and a scion. The economics of apple
production has encouraged the use of dwarfing
rootstocks for raising high density orchards1.
Historically, dwarfing rootstocks of clonal origin have
received much attention in European countries with
regard to their development and use2. Today, much of
commercial apple production in US and Europe is on
dwarfing rootstocks, because high tree density allows
greater early production, an earlier return on capital
investment, sustained high yields of good quality
fruit, lower pesticide requirement and higher labour
efficiency3-5.
Apple has wider consumer acceptability in India
both as a fresh table fruit and value added processed
products. Its cultivation is mainly restricted to
temperate Himalayan regions of Jammu and Kashmir,
Himachal Pradesh and Uttaranchal with an average
productivity of 5.68 t/ha. Among the Himalayan
states, Jammu and Kashmir has emerged as the largest
apple producing region with a productivity of 10.09
t/ha, which again is 3 to 4-fold lower compared to
horticulturally advanced countries where apple
orchards are raised predominantly on dwarfing
rootstocks6. The main bottleneck for adoption of high
density plantation in the temperate Himalayan States
of India, wherever feasible, is the limited availability
of clonally propagated dwarfing rootstocks.
——————
*Author for correspondence:
Tel.: 91-194-2461258, Ext. 264; Mobile : 09419010498; Fax: 91194-2463255
E-mail : [email protected]
Abbreviations: BAP-6-benzyl amino purine; IBA-indole-3-acetic
acid; Kn-kinetin; MS-Murashige & Skoog medium; PGR-plant
growth regulator
Conventional stool bed layering technique of
propagation7, being season-bound, irregular and slow,
is not appropriate to meet ever increasing huge
demand of dwarfing stocks, which by conservative
estimate is 1.5 million plants per year in Jammu and
Kashmir state alone6. However, the micropropagation
544
INDIAN J BIOTECHNOL, OCTOBER 2006
methods provide an efficient and alterative way for
the production of quality propagules for high density
orchards8,9. There have been several reports on
micropropagation of apple rootstock10,11; however,
results have been variable with continuing limitations
in establishing initiating cultures as the initial shoot
tip explants were difficult to establish in vitro12.
Micropropagation of apple rootstocks using bioreactor
has been recently attempted with enhanced efficiency,
but this approach has been found to be associated with
some incidence of hyperhydricity13. From the stand
point of successful micropropagation, in vitro
initiating culture system and culture proliferation are
equally important. There is, however, a tendency,
especially in woody tree species, to lay relatively
more emphasis on repeated proliferation by sustained
subculture12,14. This situation can lead to over
dependence of sustained subcultures of proliferating
material for plantlet production with either no or
occasional recourse to initiating cultures originating
from primary explants. While continued subculture
may also be associated with rejuvenation of shoots15,
but at the same time there is an attendant risk of
inadvertent selection of fast growing somaclonal
shoots12, which may impinge on genetic fidelity.
Earlier, our laboratory has reported an efficient and
reproducible in vitro initiating culture system, mutatis
mutandis, for apple16,17, which was initially proposed
for grapes18 while using explants derived form forced
stock plants. These structured efforts were later
followed by attempts to develop a protocol for
complete plantlet production of dwarfing apple
rootstock, which is reported in the present
communication.
Materials and Methods
Preparation of Explant Source
Stool beds of mature (15-year-old) stock plants of
M9 apple root stock were selected from consolidated
block in open field in the Division of Pomology at the
main University Campus, Shalimar, Srinagar. Pruned
wood was collected from the selected plants at the
inception of dormancy during November-December.
The dormant cuttings were subjected to forcing
treatment, first proposed by Dalal et al for grapes18
and later adopted for apple16. For forcing, dormant
cuttings (terminal or subterminal) of l5-20 cm length
(10-15 mm diam) were collected from mature stool
beds, washed in running tap water, pretreated with
0.2% Captan (Captan 50 WP) and stored in cold store
at 4±3°C in polythene bags until use. After meeting
the requisite chilling units of at least 42 d, the cuttings
were withdrawn from the cold store and the basal
ends of the stems were re-cut by about l cm and
placed in glass jars containing distilled water. The
amount of distilled water in the jar was kept sufficient
to cover 5 cm of the basal portion of the cuttings (Figs
1.1a-c). The cuttings were incubated in growth
chamber at 24±1°C for under 16/8 h photoperiod with
40±3 µmol m-2 s-1 light intensity. The water in glass
jar was changed every 3 to 4 d using fresh distilled
water. The number of buds on the prepared cuttings
ranged from 5 to 8. In course of the incubation, the
cuttings sprouted and rooted with varying propensity.
Shoots put forth by the sprouted buds served as
explant source from the forced stock plants. A crop of
forced shoot tip explants became ready for harvesting
within l5±5 d (Fig. 1.1c). After harvesting first crop
of forced explants (Fig. 1.2), the cuttings were again
ready for harvesting of second crop of explants within
7±2 d (Fig. 1.3).
Explant Preparation and Surface Sterilization
Shoot tips (10-20 mm in length) harvested from
forced stocks were collected into wide mouthed 250
mL flasks containing distilled water. Following the
removal of as many small leaves as possible without
the aid of microscope, explants were washed under
running Aquagaurd (Hi-flo) filtered and purified tap
water for 10 min. One to two drops of Tween-20 was
added to the explants immersed in tap water and, after
vigorous shaking, the explants were again washed
thoroughly in the running tap water for another 10
min. Washed explants were successively surface
sterilized in 75% (v/v) alcohol (30 sec), 0.1% (w/v)
mercuric chloride plus 2 drops of Tween-20 per 100
mL disinfectant solution (10 min) and rinsed 4 to 5
times in sterilized (autoclaved) double distilled water.
Shoot tips comprising the apical bud and one to two
nodes with its expanded leaves removed, were
excised aseptically and inoculated on establishment
medium of initiating cultures.
Composition of Media, Sterilization and Incubation of
Cultures
Primary explants were established following a
slightly modified version of culture initiation
procedure developed earlier17 in our laboratory. For
further growth and proliferation, two pathways were
followed. The surviving explants in the initiating
DALAL et al: IN VITRO CLONING OF APPLE
cultures were transferred in the multiplication
medium after 5±1 week. The multiplication medium
was same as initiating MS basal medium19, but
containing different concentration of growth
regulators as supplements (Table 1). For each
treatment 5 rimless culture tubes (150 mm×25 mm),
containing one explant per tube, were taken and each
experiment was repeated 4 times. Observations were
recorded after every 4±1 week. After establishing
culture conditions for optimum proliferation (Fig.
1.4), another pathway of shoot multiplication was also
followed in which proliferating material was
repeatedly subcultured on fresh but suitably PGR
amended medium (Table 2). On the first transfer, a
cluster of shoots including mother explant was
subcultured. Subsequently, the micro shoots (Fig. 1.5)
as well as the basal portion of the clump (Fig. 1.6)
that remained after harvesting the in vitro generated
shoots were sub-cultured in repeated cycles for further
multiplication. Nodal segments 5 to 15 mm in length,
obtained from in vitro raised micro shoots, were also
used to initiate proliferating cultures.
For initiation of in vitro rooting, well developed
shoots measuring 15 to 20 mm in length were excised
from cultures of proliferating shoots and inoculated
on full and half strength basal MS medium variously
supplemented with different concentration of IBA
(Table 3). After exposure to root initiating medium
for 8±3 d, the microshoots were reimplanted on
auxin-free half strength MS medium containing either
half or full concentration of organics for root
development. For each treatment of root initiation, 20
to 30 shoots were accommodated per flask (150-250
mL) and replicated five times.
The pH of all media was adjusted to 5.7 with 1.0 N
NaOH/HCl before adding agar (6 g L-1) into them.
The hot aliquots were dispensed in culture vessels
plugged with non-absorbent cotton and autoclaved
(1.06 kg cm-2, 121°C) for 15 min. The primary
explants inoculated on the MS initiation medium
(supplemented with BAP 2.2 µM + 1BA 0.49 µM)
were subjected to three-step culture initiation
procedure. Accordingly, cultures were first exposed
to refrigeration temperature (4±3°C) for 2 to 3 d
under complete darkness. The refrigerated
cultures were transferred to growth chamber for
exposure to normal-culture incubation conditions
(16/8 h photoperiod, 24±1°C temperature and 40±3
µmol m-2 s-1 light intensity, using 40 W Philips
fluorescent tubes) for 18±6 h and then followed
545
by single sub culture on fresh medium of the
same composition. The cultures on fresh medium
were incubated under normal-culture incubation
conditions with 70±5% relative humidity in a
culture room. All other subsequent cultures were
incubated under the normal-culture incubation
conditions.
Hardening of Micropropagated Plantlets
Hardening of micropropagated plantlets was done
by gradually exposing the plantlets from a highly
saturated humid condition inside the flask to a
relatively low humid ambience (70±5% RH) of the
growth chamber. This was achieved by gradually
loosening and removing the cotton plugs over a
period of 1 week. During this period, the plantelets
continued to remain healthy with turgid leaves. They
were deflasked and washed in running tap water to
remove adhered agar, followed by drenching in 0.1%
(w/v) Bavistin (BASF India Ltd.) and carefully
transplanting to polyvinyl cups or polybags,
containing pre-autoclaved sand, garden soil and
compost mixture (1:1:1; v/v) moistened with onefourth strength of MS basal salt solution. The plantlets
were housed in a shaded zero-energy polyhouse,
maintaining gradually decreasing humidity regime
(95% RH to 70±5% RH) over a period of 5±1 weeks.
After first fortnight of ex vitro growth, the plants were
subjected to foliar feeding with dilute (×1/4) MS basal
salt solution.
Results and Discussion
Proliferation of shoots was observed in all the
initial shoot multiplication MS media with various
combination regimes of PGRs (Table 1), where BAP
being common in all combinations. Superiority of
BAP in inducing axillary branching or shoot bud
differentiation is established particularly in case of
woody plants20,21. A remarkable augmentation of
multiple shoot formation (5-7 shoots) by axillary
branching was observed in MS medium supplemented
with 2.22 µM BAP, 0.49 µM IBA and 2.32 µM Kn
(Fig. 1.4). The synergism obtained in combination of
two cytokinins was similar to that reported for auxins.
Synergism between two or more cytokinins has also
been reported for apple and other plant species in
respect of shoot proliferation24,25. About 4-fold
multiplication rate (Table 2) was achieved on the
same medium with amended PGR regime, containing
halved cytokinin concentration, while adopting
another pathway of shoot multiplication. In this
INDIAN J BIOTECHNOL, OCTOBER 2006
,.,..
Fig. I-In vitro cloning of M 9 apple rootstock: 1.1, Forcing of pre-chilled apple cuttings in growth charnber:(a) immediately after
withdrawal from cold store; (b) initiation of sprouting after I week; and (c) shoot tip explants ready for harvesting after 2 weeks. 1.2,
Cuttings after harvesting first crop of forced explants. 1.3, Forced cuttings ready for harvesting of second crop of explants. 1.4, Shoot
proliferation of primary explants on multiplic~ationmedium. 1.5-1.7, Shoot proliferation in course of repeat subculture of proliferating
material: 1.5, proliferating microshoots; 1.6, subcultured basal sectored clump; and 1.7, proliferating basal sectored clump. 1.8, 3kl week
old proliferating cultures ready for repeat subculture. 1.9, In vitro rooting of microshoots in root development medium. 1.10, Hardened
plantlets grown ex vitro.
DALAL et al: IN VITRO CLONING OF APPLE
547
Table 1—Effect of different concentration of cytokinins (Kn and BAP) and auxin (IBA) on multiple shoot induction from established
shoot tip explants of M. domestica Borkh transferred to multiplication MS medium
PGRs (μM)
IBA
Kn
0.00
0.49
0.98
0.00
0.49
0.98
0.00
0.00
0.00
2.32
2.32
2.32
Response
BAP (1.11 μM)
Shoot
% sprouted
number
±SD
45.0
2.0±0.42
35.0
3.0±0.48
35.0
3.5±0.51
40.0
3.0±0.45
75.0
5.0±0.82
40.0
3.5±0.58
Table 2—Effect of repeat subculturing on shoot multiplication
and elongation of M. domestica Borkh on fresh multiplication MS
medium every 3±1 weeks
Repeat
subculture
First
Second
Third
BM1*
BM2
Shoot
Shoot
Shoot
Shoot length
number
number
length
(mm)±SD
±SD
±SD
(mm)±SD
18.2±2.5
20.5±5.4
25.1±2.9
26.5±4.8
70.5±9.3
22.3±5.2 110.0±11.1 28.3±5.3
230.6±30.1 25.9±4.9 501.0±35.6 29.9±4.5
*BM1 = (MS + BAP 2.22 μM + IBA 0.49 μM + Kn 2.32 μM)
BM2 = (MS + BAP 1.11 μM + IBA 0.49 μM + Kn 1.16 μM)
pathway,
proliferating
material
comprising
microshoots, nodal segments and sectored basal
portion of proliferated clumps (Figs 1.5-1.7) were
repeatedly subcultured. Proliferating cultures would
be ready for repeat subculture for every 3±1 week
(Fig. 1.8). Apart from higher shoot yield in this
process, shoots were also vigorous and longer than
those obtained during initiation of cultures on shoot
multiplication medium. Shoot cultures have been
maintained by repeated subculture for as long as 36
months without loss of vigour or change in the rate of
multiplication in other tree species14, whereas in apple
even nine year old culture lines have been maintained
by sustained subculture without adverse effect on
shoot production and adventitious rooting12.
Transfer of shoots from multiplication phase onto a
medium with altered nutritional or phytohormonal
levels has been emphasized for stem elongation to
facilitate rooting26. There is also a need to strike a
balance between use of primary cultures and
proliferating material by repeated subculture for shoot
multiplication without risking the adverse impact on
genetic fidelity, as the aggressive and accelerated
multiplication regime of repeated subcultures are
Shoot
length
(mm)±SD
12.8±3.9
13.5±3.2
16.0±4.1
14.2±3.8
16.2±4.8
16.0±4.5
BAP (2.22 μ M)
Shoot
% sprouted
number
±SD
40.0
2.5±0.45
55.0
3.5±0.52
40.0
3.0±0.48
30.0
2.5±0.41
85.0
6.8±0.80
65.0
4.0±0.65
Shoot
length
(mm)±SD
17.5±4.6
10.3±2.9
13.8±3.1
15.2±3.5
15.5±5.1
14.2±4.9
fraught with inadvertent selection of fast growing
somaclonal shoots12 or rejuvenation associated with
micropropagation15. Although shoot multiplication
was achieved by genetically reliable pathway of
axillary branching without intervening callus phase,
somaclonal shoots could arise due to occurrence of
occasional adventitious shoots and/or rare mutational
events. In spite of this, there is a tendency to heavily
bank upon the pathway of repeated subcultures of
proliferating material for large-scale production of
micropropagated plantlets. This situation is generally
encountered in various adult and woody tree species,
such as apple, where initial shoot tip explants in the
primary cultures are difficult to establish in vitro12. In
the proposed protocol (Fig. 2), this limitation was
overcome by using explants derived from forced stock
plants in accordance with the procedure developed
earlier in this laboratory16,17.
Young shoots, when subjected to root initiating
treatment, invariably callused at the base and over a
period of time callus mass increased and root initials
were barely visible. A two-step strategy for in vitro
rooting was adopted. In the first step micro shoots
were implanted in basal MS medium supplemented
with various IBA concentrations. Maximum root
initiation and least callusing were observed on full
strength MS medium supplemented with 14.70 µM
IBA (Table 3). Exposure to root initiation medium
was not allowed for more than 11 d, since
microshoots were overwhelmed by callus mass and
root development was arrested. Indeed, root
development was apparent in course of as brief as 5 d
exposure of microshoots to rooting medium. In the
second step, immediately after root initiation, the
microshoots were reimplanted on PGR free half
strength MS medium for root development (Fig. 1.9).
The root formation was maximal when organics were
548
INDIAN J BIOTECHNOL, OCTOBER 2006
Fig. 2—Protocol for micropropagation of M9 apple rootstock using explants derived from forced stocks: a. Forcing of stock plants; b.
Three-step culture initiation; c. Two cultural pathways of shoot multiplication; d. Two-step in vitro rooting; and e. Hardening of plantlets.
DALAL et al: IN VITRO CLONING OF APPLE
549
Table 3—Rooting response of microshoots of M. domestica Borkh as influenced by IBA concentration and root development response of
root initiated plantlets reimplanted on PGR free half strength MS medium
Strength of
MS root
IBA
initiating
concentration
media
(μM)
(minerals +
sucrose)
X0.5
X1.0
7.35
14.70
29.40
7.35
14.70
7.35
Root initiation responsea
MS organic concentration
X0.5
X1.0
Rooting
Rooting
Callus
Callus
initiation
initiation
intensity
intensity
(%)
(%)
0.0
20.8
10.0
0.0
75.9
35.0
+
+
+++
+
+
+++
0.0
2.9
20.8
0.0
95.3
40.0
++
+
+++
+
+
+++
Root development responseb
MS organic concentration
X0.5
X1.0
Root number Root length Root number Root length
±SD
(mm) ±SD
±SD
(mm) ±SD
1.5±0.16
1.8±0.21
8.5±1.28
2.2±0.31
21.5±5.51
18.2±4.91
28.0±3.79
19.5±4.00
1.8±0.21
2.0±0.28
9.2±0.95
4.5±0.42
23.2±3.51
21.5±4.00
35.0±4.44
25.5±4.21
a : Recorded after 8±5 d; b : Recorded after 4±1 week
supplied at full strength. The promotory effect of
reduced salt concentration of basal medium on in
vitro rooting of shoots has earlier been reported14,27.
None of the in vitro plantlets transferred directly to
pot mix, survived under ex vitro conditions. However,
the survival percentage rose appreciably when they
were subjected to in vitro hardening before being
transferred to polyvinyl cups or polybags (Fig. 1.10).
Thorough washing of plantlets followed by
carbendazim drenching reduced incidence of disease
and wilting. Carefully controlled regime of relative
humidity which was gradually brought down from
95% to 70±5% over a period of 5±1 week resulted in
80% survival. Again, prevention of plantlets from
desiccation during initial 7 d ex vitro growth was
crucial for successful hardening. Initial moistening of
pot mix with mineral salts and subsequent foliar
feeding resulted in improved vigour of plants.
The procedure described in the present
investigation proposes a novel strategy for large-scale
propagation of dwarfing apple rootstocks. The
strategy encompasses, among other things, use of
forced primary explants in a three-step culture
initiation procedure for development of successful
protocol for apple micropropagation (Fig. 2).
Acknowledgement
Authors thank the Indian Council of Agricultural
Research, New Delhi, for part-financial support to the
research programme. Thanks are also due to Dr
Charanjit Kaur Sahni for going through the
manuscript
critically
and
making
valuable
suggestions, and Bashir Ahmed Mir for his help and
cooperation in the course of investigation.
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