2014 STUDIES ON IN VITRO CLONING OF MATURE TREE OF CAPPARIS DECIDUA AND BOSWELLIA SERRATA
2.
REVIEW OF LITERATURE
Plant biotechnology having tissue culture as an important tool offers
many approaches for genetic improvement of any plant species. Among
these, micropropagation is a powerful tool for fast multiplication of selected
genotypes at faster rates; therefore, it has been adopted in agriculture as well
as in forestry (Bajaj, 1986).
The history of plant tissue culture begins in 1838-1839 when
Schleiden (1838) and Schwann (1839) independently stated the ‘Cell
theory’ and proposed the ‘totipotency’, which state that cells are
autonomic, and in principle, are capable of regenerating to give a
complete plant. In 1878, Vochting reported callus development in
numerous species. Based on this premise, in 1902, a German physiologist,
Gottlieb Haberlandt developed the concept of in vitro cell culture. He cultured
single cells from different plant species like palisade cells from leaves of
Laminum purpureum, glandular hair of Pulmonaria and pith cells from petioles
of Eicchornia crassiples etc in Knop’s salt solution enriched with sucrose,
asparagine and peptone. In his cultures, cells increased in size, accumulated
starch but failed to divide. Despite lack of success, Haberlandt made several
predictions about the requirements in media in experimental conditions
which could possibly induce cell division, proliferation and embryo
this period, an innovative approach to tissue culture using meristematic cells
like root and stem tips was reported by Kotte (1922) and Robbins (1922)
working independently.
A significant breakthrough in tissue culture was the discovery of
auxin by Went (1926) and its characterization by Koegel et al. (1934).
This led to the possibility of culturing plant tissues for indefinite periods,
reported by White (1939), Nobecourt (1938, 1939) and Gautheret (1939).
Chapter 2: Review of Literature
induction. G. Haberlandt is thus regarded as father of tissue culture. Within
20
2014 STUDIES ON IN VITRO CLONING OF MATURE TREE OF CAPPARIS DECIDUA AND BOSWELLIA SERRATA
However, organogenesis became feasible only after the discovery of another
growth regulator, the cytokinins. Upto this time the main objective of
tissue culture work was ascertaining the possibility of culturing the cell
indefinitely.
The starting point of modern in vitro vegetaitive propagation
methods (micropropagation) were started with Ball (1946), as he pointed
out exactly which part of a shoot meristem gave rise to a whole plant.
Tissue culture was utilized for eradication of viruses through meristem
culture (Morel and Martin, 1952), culture of single cells and suspension
cultures (Muir et al., 1954), establishment of auxin-cytokinin basis of
organogenesis (Skoog and Miller, 1957), somatic embryogenesis (Reinert,
1958) and large-scale culture of cells (Tulecke and Nickell, 1959). The
sixth decade of the last century was a story of success in tissue culture
with successful isolation of plant protoplasts (Cocking, 1960). Reports
concerning the recovery of plants from haploid cells were obtained with
Datura innoxia (Guha and Maheshwari, 1964). The isolated protoplasts,
under culture conditions are known, to form colonies (Kao et al., 1971),
to form cell walls (Prat, 1972) and to regenerate into plants (Nagata and
Takebe, 1971; Schuma and Koblitz, 1983).
The history of plant tissue culture and its applications have been
reviewed and discussed from time to time (Gautheret, 1983, 1985; Krikorian,
of using tissue culture in micropropagation and biotechnology related to
forestry improvement have been critically and elegantly analyzed in several
reviews (Thorpe and Biondi, 1984; Dunstan and Thorpe, 1986; Bonga and
Durzan, 1987; Haissing et al., 1987; Thorpe, 1991; Mascarenhas and
Murlidharan, 1989 and Hammatt, 1992).
Though in the initial stages of growth of plant tissue culture as a
Chapter 2: Review of Literature
1988; Thorpe, 1990; Gamborg, 2002; Vasil, 2008). The problems and potentials
discipline was very slow, gradually it achieved momentum and has seen very
21
2014 STUDIES ON IN VITRO CLONING OF MATURE TREE OF CAPPARIS DECIDUA AND BOSWELLIA SERRATA
fast developments. This has resulted in a transfer of attention from basic
research to applications. Virus eradication, micropropagation, production of
secondary products, production and uses of somaclonal variations, somatic
embryogenesis, haploid production and diploidization, embryo rescue,
protoplast culture, regeneration and fusion to produce somatic hybrids, gene
transfer etc. are some of the areas of plant tissue culture which have applied
significance. The most widely used application of the tissue culture
technology in agriculture, horticulture and forestry is the clonal propagation
of plants. Propagation through tissue culture is popularly referred as
micropropagation. Micropropagation can be aptly defined as vegetative
propagation under controlled and aseptic conditions in the microenvironment of the culture vessel, which mimics the growth requirements of
a plant in the natural conditions. Propagation of plants can be achieved
through three regeneration pathways, viz. axillary bud proliferation, somatic
embryogenesis and adventitious bud formation (Murashige, 1974, 1977).
Micropropagation through axillary bud proliferation where no
intermediary callus is involved can give rise to true to type plant population
and is the safest and the most widely used method to maintain clonal fidelity.
Over the years, a variety of plant species have been successfully
micropropagated using nodes with pre-formed buds in axils from juvenile as
well as mature sources. Axillary shoots proliferating from these pre-formed
and hence selected superior stock with desired traits can be multiplied on a
mass scale for increasing the productivity, utilizing this propagation strategy.
These axillary and fascicular buds proliferate into shoots when cultured on
media supplemented with different hormonal combinations.
Sufficient literature is available on various aspect of tissue culture
studies on medicinally important plant and desert trees.
Chapter 2: Review of Literature
meristems have an advantage of being the true compliment of explant source
22
2014 STUDIES ON IN VITRO CLONING OF MATURE TREE OF CAPPARIS DECIDUA AND BOSWELLIA SERRATA
Mathur and Chandra (1983) and Nandwani and Ramawat (1991)
document promontory role of cytokinins with auxins on axillary bud
proliferation of Acacia nilotica and Prosopis cinneria respectively. Auxin were
reported integral for shoot differentiation on cultured nodes of both the
species.
Rohini and Gupta (2002) reported multiple shoot initiation in stem
nodes excised from in vitro grown seedlings of Acacia catechu, on MS medium
supplemented with 1 to 100 µM of BA. Subculturing was done on the same
medium supplemented with 1.5 g/l of polyvinylpyrrolidone (PVP). In the
second subculture, 9-10 shoots differentiated per explant when transferred to
a medium lacking PVP, containing 10 µM of BA. If individual shoots along
with some callus were subcultured on BA (10 µM), nearly 15 shoots per
explant regenerated in 90 days. Rooting was observed on ½ strength MS
medium with 14.7 µM of IBA. 40 mg/l of glutamic acid prevented leaf
senescence in the rooting medium. Garden soil, sand and silica (1:1:1) found
suitable for hardening.
Robinson et al. (2005) selected 20 year old tree of Tecomella undulata and
harvested explants during March-May. The most effective media for
maximum regeneration (95 %) of nodal explants was MS + 1.5 mg/l BA + 0.02
mg/l IAA. Nodal segments were found best as compared to shoot tip and
internodal segment explants. The cultures were kept at 25 ± 1 oC, 2000 lux of
exposed to 16/8 h cycle of light and dark. Twenty nine shoots per nodal
segment explant were observed on MS + 0.75 mg/l BA + 0.01 mg/l IAA
within 3 weeks. 66 % of rooting was recorded by the two step procedure. In
the first step, a 48 hr. treatment of ½ MS liquid medium + 2.5 mg/l IBA and in
the second step these shoots were transferred to ½ MS basal medium. 73 % of
plantlets survived in potting mixture composed of drained soil + vermiculite
(3:1).
Chapter 2: Review of Literature
light from fluorescent tubes and 60-70 % relative humidity. The cultures were
23
2014 STUDIES ON IN VITRO CLONING OF MATURE TREE OF CAPPARIS DECIDUA AND BOSWELLIA SERRATA
Gyes et al. (2007) established a protocol for in vitro micropropagation
from nodal explants of Tectona grandis. Multiple shoots were obtained on
modified MS medium supplemented with 1.5 mg/l BAP, 0.01 mg/l IBA and
0.1 mg/l GA3. 100% of shoots were rooted on modified MS medium
containing 0.5mg/l IBA and 160 mg/l putrescene. In vitro regenerated
plantlets were transferred in green house where they survived at 100%.
Rathore et al. (2007) reported in vitro cloning of mature tree of Citrus
limon using nodal shoot explants on MS medium supplemented with 9µM
BAP. 3 to 4 multiplication rate was achieved on MS medium having ½
NH4NO3, KNO3 and (NH4)2SO4 with 0.22µM BAP. 100% shoots were rooted
on ½ strength MS medium supplemented with 27 µM NAA+ 0.1% activated
charcoal and ex vitro rooting on solirite pulse treated with 1.07-2.68µM NAA.
95% of rooted plantlets survived hardening and transplanting.
Khalfalla and Daffalla (2008) reported in vitro multiplication of Acacia
senegal. Multiple shoots were regenerated from cotyledonary node and nodal
segment on MS medium supplemented 1.0 mg BAP. 8.3±0.3 shoots per
cotyledonary node and 5.3±0.7 shoots per node explant were obtained. 25%
shoots were rooted on MS medium supplemented with 1.0 mg/l IBA.
Srivastava et al. (2009) reported maximum rooting and establishment
percentage (87.66%) in Kiwi plant (Actinidia chinensis) cuttings treated with
roots per cutting (52.5) and length of the longest root (21.53 cm) compared to
control (15 and 6.5 cm, respectively). Field establishment was also higher
(98%) in case of IBA compared to control (42.5%).
Ismail et al. (2011) established in vitro propagation system for direct
shoot regeneration in Clitoria ternatea L. using different explants, viz., nodal,
cotyledonary node and shoot tip. The highest mean no. of shoots (18.7±1.0)
were obtained on MS medium supplemented with BA (5 µM) at pH 5.8. In
Chapter 2: Review of Literature
IBA at 5000 ppm against control (12%). They also got maximum number of
24
2014 STUDIES ON IN VITRO CLONING OF MATURE TREE OF CAPPARIS DECIDUA AND BOSWELLIA SERRATA
vitro regenerated shoots were rooted on ½ strength MS medium fortified with
2 µM IBA. Complete plantlets were transplanted to natural condition where
they exhibited 80% survivability.
Phulwaria et al. (2011) have selected some of the trees of S. persica from
the branches of the middle trunk region and were lopped during Dec-Jan and
June-July and explant was collected during March-April and July-September.
80 % of the explants showed bud break when harvested during the months of
Dec-Jan and June-July from lopped portion of the tree. 90 % of the nodal
explants produced on MS + 8.88 µM BA with 3.1 shoot per node. The shoots
were repeatedly subcultured on MS + 4.44 µM BA for shoot multiplication.
Among different concentration of NAA & IAA tried, 1.11 µM BA + 1.16 µM
Kn + 0.54 µM NAA was found best for shoot multiplication. The shoots were
pulse treated with 2460.27 µM of IBA + 494.56 µM of NOA exhibited 90 %
rooting.
Parmar et al., (2012) reported micropropagation and evalvuation of
genetic fidelity of Combhiphora wightii. Nodal segements were inoculated on
MS medium supplemented with 8.88 µM BAP + 0.57 µM IAA and additives
(50mg/l ascorbic acid + 25 mg/l citric acid + 25 mg/l adenine sulphate).
Shoots were multiplied on same medium. Shoots were pulse treated on liquid
medium supplemented with 4.92 µM IBA + 5.71 µM IAA for 24 hrs under
dark condition, followed by transfer to semi-solid ½ strength MS medium
after 4-5 week .In vitro hardening step the survival was 61.5% and ex vitro
hardening step it was 100%. In genetic fidelity test uniform banding pattern
was observed in all tissue culture raised plants without any polymorphism.
Phulwaria et al. (2012) established a micropropagation protocol for
large scale multiplication of Celastrus paniculatus. Multiple shoot induction
was observed on MS medium supplemented with 2.0 mg/l BAP. MS medium
Chapter 2: Review of Literature
with 2% sucrose and 0.5% activated charcoal. 86.7% rooting was achieved
supplemented with 0.5 mg/l BAP and 0.1 mg/l IAA was found to be best for
25
2014 STUDIES ON IN VITRO CLONING OF MATURE TREE OF CAPPARIS DECIDUA AND BOSWELLIA SERRATA
shoot multiplication. 100% rooting was observed when shoots were pulse
treated with 300 mg/l IBA for 3 min. 95% plantlets were acclimatized
successfully.
Rodrigues et al. (2012) reported in vitro propagation of Azadiracta indica.
Nodal explants were inoculated on MS medium supplemented with 0.5 mg/l
BA, 0.5 mg/l Kn and 0.05 mg/l NAA. When flasks were sealed with two
polytetrafluroethylene hydrophobic membranes (PTEE), highest no. of shoot
were produced in contrast to cultures in flask without membrane where
cultures showed leaf chlorosis and senescence. For plant recovery and
regenerates were acclimatized in a substrate of coconut fiber and eucalyptus
bark (1:1) and showed 80% survival.
Ujjwala et al. (2013) reported clonal propagation of Gmelina arborea
through stem cutting. The stem cuttings were showed highest rooting
percentage response at 1000ppm NAA treatment. More number of nodes
were sprouted at 2000 ppm NAA comparison to other concentration of stem
cutting.
Raghavaswamy et al. (1992) cultured nodal explants from mature tree
of Dalbergia latifolia on MS medium containing a combination of BAP with any
of the three auxins (NAA, IAA and IBA). Of these combinations, medium
supplemented with BAP and NAA was more efficient for axillary bud break.
(summer, monsoon, autumn and spring) and NAA affect the rooting
behaviour of culm cuttings of Bambusa balcooa.the finding showed that
sprouting percent (53.18%), rooting percent(28.29), number of roots (9.19) and
length of roots (12.73) was best in spring season under the intermitted
conditions.
Singh et al. (2012) reported that different concentration of auxin (IBA,
IAA and NAA) affected the rooting in Crataegus crenulata cuttings. The results
Chapter 2: Review of Literature
Joshi et al (2012) reported that age, growing conditions, season
26
2014 STUDIES ON IN VITRO CLONING OF MATURE TREE OF CAPPARIS DECIDUA AND BOSWELLIA SERRATA
showed that rooting percent (81%), sprouting percent (89%) and root length
(18.12) was achieved when cuttings were treated with IBA 250ppm.IBA
(250ppm) treated cutting were found to be best maximum plant length
(189.32cm) and number of branches (6.45) after six month of field plantation.
Janifer et al (2013) concluded that exogenous hormone treatment
improve rooting ability of Hippophase rhamnoider. Rooting percent was
maximum in (85.50%), number of branch (18.67) and number of roots (16.67)
was observed in hard wood cuttings were treated with IBA 300ppm for 48
hour dip treatment.
2.1
Review of Capparis decidua
Deora and Shekhawat (1995) developed a microprpagation protocol of
Capparis decidua on MS medium supplemented with 5.0 mg/l BAP + 0.1 mg/l
NAA + additives (50mg/l Ascorbic acid, 25mg/l adenine sulphate, 25mg/l
citric acid, 25mg/l L-arginie). The shoots were multiplied on MS medium
supplemented with 1.0 mg/l BAP +0.1 mg/l IAA + additives. 60-70% of the
shoots were rooted when pulse treated with 100mg/l IBA in half strength MS
liquid medium for 4 hours and then transferred on hormone free half strength
agar gelled MS medium. Tissue culture raised plants were hardened and
transferred to field where 75% survival was obtained.
Tyagi and kothari (1997) reported in vitro clonal propagation of
cotylidon and hypocotyl explant taken from the seedling. MS medium
supplemented with 5mg/l BAP was found to be best for shoot bud
proliferation from the nodal as well as seedling explant. Shoot multiplication
was best on cotylidonary node. They also obtained multiple shoots from
nodal segments of mature tree of Capparis decidua on MS medium
supplemented with 3.0 mg/l BAP. 65% rooting was achieved on MS medium
supplemented with 1.0 mg/l IBA.
Chapter 2: Review of Literature
Capparis decidua using nodal explant from mature tree and cotyldonary node,
27
2014 STUDIES ON IN VITRO CLONING OF MATURE TREE OF CAPPARIS DECIDUA AND BOSWELLIA SERRATA
Tyagi et al. (2005) developed an efficient protocol for direct somatic
embryogenesis in Capparis decidua. They cultured mature zygotic mature
embryos on MS medium supplemented with 2,4-D (0.1 mg/l) and BA (0.5
mg/l). Somatic embryos were developed without intervening callus phase
from the sub epidermal cells. Treatment with ABA promoted maturation of
somatic embryos and BA (1 mg/l) promoted germination.
Chalak and Elbitar (2006) developed a protocol for in vitro propagation
of Capparis spinosa. Multiple shoots were obtained on MS medium
supplemented with 1mg/l Zeatin. High rooting response of shoots (92%) was
obtained after 4hrs pulse treatment period in darkness with 100 mg/l IAA
solution, followed by culture on half strength MS basal medium. These results
indicate the enormous potential of Capparis spinosa subsp rupestris that could
be used for large scale multiplication.
Sexena et al. (2007) developed a micropropagation protocol for multiple
shoot regeneration employing shoot tip explants of Capparis decidua. Shoot
induction was observed on MS medium supplemented with 7.0 mg/l BAP
and 0.1 mg/l NAA where an average 8.5 shoots per explants were obtained.
Regenerated shoots were rooted best on MS medium supplemented with 1.0
mg/l IBA. Rooted shoots were hardened and transferred to field with 83%
survival.
Capparis decidua using nodes and shoot tip explants. Maximum shoot
induction response was obtained on MS medium supplemented with 5.0
mg/l BAP +25.0mg/l adenine sulphate, 3-4 shoots proliferated from nodal
explants. 37% rooting response was observed on MS medium with 1.0 mg/l
IBA and 1.0 mg/l NAA.
Chalak et al. (2001) developed a protocol for in vitro propagation
of Capparis spinosa via seeds and nodal buds of a Lebanese ecotype. High seed
Chapter 2: Review of Literature
Chahar et al. (2010) standardized a protocol for clonal propagation of
28
2014 STUDIES ON IN VITRO CLONING OF MATURE TREE OF CAPPARIS DECIDUA AND BOSWELLIA SERRATA
germination percentage (71%) was achieved on MS medium deprived of
hormones. Multiple shoots were obtained from nodal buds on both MS and
modified MS medium supplemented with BAP (1.5 mg/l), IBA (0.05 mg/l) ,
GA3 (0.1 mg/l). In vitro shoots were multiplied on the same medium. 87%
rooting was obtained after a 4hrs pulse treatment in darkness with IAA (100
mg/l), followed by a subsequent 30 days of culture on gelled half-strength MS
basal medium. These result indicate the enormous potential for caper to be
used for large scale potential.
Basu et al. (2009) developed a protocol for in vitro propagation by
multiple shoot induction of Crataeva religiosa , a medicinal tree having high
medicinal values belonging to the family Capparidaceae. High frequencies of
multiple shoot regeneration were achieved from apical bud on MS medium
fortified with 8 mg/l BAP alone. Five to seven shoots per explants were
obtained. The elongated shoots were sub cultured for rooting on half strength
MS medium supplemented with various concentrations of IBA and IAA.
The in vitro raised plantlets were acclimatized in green house and successfully
transplanted to natural condition with 72% survival.
Musallam
et
al.
(2011)
developed
a
protocol
for
in
vitro
micropropagation of Capparis spinosa. Many media were tested; MS medium
(Murashige and Skoog) were tested at different basal levels, modified MS
medium (½MSD) and WPM (Lloyd and McCown) for the establishment
Multiple shoots were obtained on WPM medium supplemented with 0.8mg/l
kinetin in combination with 0.05 mg/l IBA and 0.1 mg/l gibberillic acid. High
frequency of rooting was obtained on ½MS media supplemented with 5mg/l
IBA. Regenerated plantlets were acclimatized with 63% survival.
Tyagi et al. (2010) developed a protocol for in vitro multiplication
of Capparis decidua from cultured leaves procured from multiplying axiliary
Chapter 2: Review of Literature
stage. WPM was found to be the best for the establishment of mother plants.
shoots on the cultured nodal explants. The highest efficiency of shoot
29
2014 STUDIES ON IN VITRO CLONING OF MATURE TREE OF CAPPARIS DECIDUA AND BOSWELLIA SERRATA
formation was observed on Murashige and Skoog (MS) medium containing 2
mg dm−3 Benzyladenine (BA) and 0.5 mg dm−3 α-Naphthaleneacetic acid
(NAA).
The
regenerated
shoots
were
transferred
to
MS
medium
supplemented with 3 mg dm−3 BA for growth and proliferation. Shoots were
transferred to MS medium supplemented with 1 mg dm-3 Indole-3-butyric
acid and 0.5 mg dm−3 Indole-3-acetic acid for in vitro rooting. No variation
was detected among the micropropagated plants by randomly amplified
polymorphic DNA (RAPD) markers.
2.2
Review of Boswellia serrata
Purohit et al. (1995) developed in vitro procedure for large scale
multiplication of Boswellia serrata using cotyledonary node segments.
Cotyledonary nodes were cultured on MS medium supplemented with 0.5
dm-3 BAP and 0.05 NAA dm-3. Shoots were multiplied on same medium.
Shoots were rooted on ¼ MS medium supplemented with 0.5 dm-3 IBA and
0.25 dm-3 IAA, 50 mg/l PVP and 100 mg/l ascorbic acid. 80% shoots were
rooted within 8-10 days. 70% plantlets were successfully established in soil :
solirite (1:1) mixture.
Ghorpade et al. (2010) established an efficient protocol for development
of seedlings of an endangered medicinally important forest tree Boswellia
serrata Roxb. Excised green zygotic embryos were cultured on Gamborg (B5)
medium. The highest frequency of embryo germination (96%) on MS medium
containing 3% sucrose supplemented with 200mg/l PVP. 94% fully developed
seedling were successfully established in soil.
Chandrashekhar
et
al.,
(2005)
developed
a
protocol
for
in
vitro multiplication of Boswellia ovalitifoliolata from coteledonary explants. The
highest shoot multiplication (7.1 fold) was achieved in 50 d on MS
supplemented with thidiazuron (2.72 µM). Excised shoots 3.0 cm were placed
Chapter 2: Review of Literature
medium, McCown and Loyad (WPM) and Schenk and Hildebrandt (SH)
30
2014 STUDIES ON IN VITRO CLONING OF MATURE TREE OF CAPPARIS DECIDUA AND BOSWELLIA SERRATA
on the MS basal medium supplemented with indole-3-acetic acid and indole3-butyric acid alone and in combinations for rooting. Activated charcoal (100
mg/l) and polyvinylpyrrolidone (40 mg/l) were added to the medium to
prevent browning of cultures. The regenerated plantlets have been
successfully acclimatized and transferred to soil.
Suthar et al., (2011) observed liquid medium devoid of agar proved to
be the best for the micropropagation of Boswellia serrata Rox.
Reduced
concentration of agar than normal and its complete elimination favored both
shoot multiplication and rooting during micropropagation of B. serrata.
Overall increase in dry and fresh wt. and chlorophyll content in such shoots.
Devi et al. (2011) achieved plant regeneration from nodal explants and
cotyledonary node explants of Sterculia urens. Multiplication shoot induction
was found to be best on MS medium supplemented with 0.90 µM TDZ.
Elongation of shoots were observed on MS medium supplemented with 8.7
µM GA3. 73% rooting was obtained on ½MS media supplemented with 19.7
µM IBA.
Suthar and Purohit (2012) observed that when micropropagated
plantlets of Boswellia serreata were biotized using an endophytic root fungus
Piriformospora indica during hardening and acclimatization stage, found
beneficial in terms of overall growth and ex vitro survival of plantlets. The
survival. Significant increase in root growth, biomass and total chlorophyll
contents in colonized plantlets.
Chapter 2: Review of Literature
fungus colonized 70% of plantlets and 75% such plantlets showed ex vitro
31