8
REVIEW OF LITERATURE
2. Botanical description of Jatropha curcas
The physic nut, by definition, is a small perennial tree or large shrub, which
can reach a height of 3 to 5 m, but can attain a height of 8 or 10 m under favourable
conditions. The plant shows articulated growth (Kumar and Sharma, 2008) straight
trunk, thick branch lets with a soft wood and a life expectancy of up to 50 years
(Achten et al., 2007). Flowering occurs during the wet season (Raju and Ezradanam,
2002) often with two flowering peaks, i.e. during summer and autumn. In the
permanently humid regions, flowering occurs throughout the year (Heller, 1996).
Flowers are unisexual, monoecious, greenish yellow colored in terminal long,
peduncled paniculate cymes. J. curcas is both monoecious and protandrous, meaning
that it cross-pollinates as well as self pollinates (Solomon and Ezradanam, 2002). The
first flowing and fruiting take place between 4 months and 2 years (Poteet, 2009), but
these fruits are either pruned off or drop off naturally. Flowering starts at the bottom
of the tree, and moves upwards. Both male and female flowers appear on the same
clusters. Per cluster, there is a 29:1 ratio of male flowers to female flowers (Poteet,
2009). The inflorescences form a bunch of green trilocular ellipsoidal fruits yielding
approximately 10 or more ovoid fruits (Tewari, 2007). The exocarp remains fleshy
until the seeds are mature, and seeds are produced in triovultate fruits that are toxic to
humans and animals due to the to toxic protein curcin as well as several other phorbol
esters that have toxic elements (Poteet, 2009). Yellow berries can be harvested, but
their oil content is lower than mature brown berries (Poteet, 2009). The process of
sowing to full harvest for J. curcas can take upwards of 2 years (Ogoshi, 2009). It is
possible to harvest low amount of fruit during the first year. The blackish seeds of
most provenances contain toxins, such as phorbol esters, curcin, trypsin inhibitors,
lectins and phytates, to such levels that the seeds, oil and seed cake are not edible
without detoxification (Makkar et al., 1997; Aregheore et al., 1998; Makkar et al.,
2007).
Review of Literature 9
2.1 Distribution and ecological requirement
J. curcas was probably distributed by Portuguese seafarers via the Cape Verde
Islands and Guinea Bissau to other countries in Africa and Asia (Heller, 1996). It is
assumes that the Portuguese brought the physic nut to Asia. It is well adapted to arid
and semi-arid conditions. J. curcas grows almost anywhere except waterlogged lands,
even on gravelly, sandy and saline soils. It can thrive on the poorest stony soil. It can
grow even in the crevices of rocks. The leaves shed during the winter months form
mulch around the base of the plant. The organic matter from shed leaves enhances
earthworm activity in the soil around the root-zone of the plants, which improves the
fertility of the soil. Regarding climate, J. curcas is found in the tropics and subtropics
and likes heat, although it does well even in lower temperatures and can withstand a
light frost. It will grow under a wide range of rainfall regimes from 250 mm to 1200
mm per annum (Katwal and Soni, 2003). In low rainfall areas and in prolonged
rainless periods, the plant sheds its leaves as a counter to drought. Its water
requirement is extremely low and it can stand long periods of drought by shedding
most of its leaves to reduce transpiration loss. J. curcas is also suitable for preventing
soil erosion and shifting of sand dunes. It grows on well-drained soil with good
aeration and is well adapted to marginal soil with low nutrient content (Openshaw,
2000). On heavy soils, root formation is reduced. J. curcas is a highly adaptable
species, but its strength as a crop comes from its ability to grow on very poor and dry
sites.
2.2 Propagation of J. curcas
2.2.1 Micropropagation
Plant micropropagation is an integrated process in which cells, tissues or
organs of aseptic environment to produce many clonal plantlets (Altman, 2000). The
technique of cloning isolated single cells in vitro demonstrated the fact that somatic
cells, under appropriated conditions, can differentiate to a whole plant. Haberlandt
(1902) first attempted to prove this theory experimentally using monocotyledonous
plants and though his attempt was unsuccessful, he elaborated the concept of
totipotency that refers to the potential of an individual cell to regenerate a whole
Review of Literature 10
plant. This potential of a cell to grow and develop a multicellular organism is termed
cellular totipotency (Razdan, 1993; Torres et al., 1999). This potential of cells or
tissues to form all cell types and regenerate a plant is the basic principle of tissue
culture. Murashige (1974) reported three stages i.e. establishment, multiplication and
rooting. Hartman and Kester (1983) added another stage i.e. acclimatization. Debergh
and Maene (1981), Debergh (1987) and Torres (1988) proposed that one more stage
should be added to the process i.e. ‘Stage O’ (Stock plant selection and its
preparation).
According to Debergh & Read (1991) and Altman (2000), the
micropropagation process can be divided in five different stages:
Phase 0: growing mother plants under hygienic conditions. It involves the production
of stock plants in greenhouse.
Phase I: initiation of culture. The purpose of this stage is to initiate axenic cultures. It
involves the selection of explants, deinfestation and the cultivation under aseptic
conditions.
Phase II: rapid regeneration and multiplication of numerous propagules
(multiplication phase). Masses of tissues are repeatedly subcultured under aseptic
conditions into new culturing media that encourage propagule proliferation. The
culture can supply shoots for the subsequent propagation phases as well as material
that is required to maintain the stock.
Phase III: elongation and root induction or development (rooting phase). This phase is
designed to induce the establishment of fully developed plantlets. It is the last period
in vitro before transferring the plantlets to ex vitro conditions.
Phase IV: transfer to ex vitro condition (acclimatization). Acclimatization is defined
as the climatic or environmental adaptation of an organism, especially a plant that has
been moved to a new environment (Kozai and Zobayed, 2000).
Significant features of in vitro propagation procedure are its enormous multiplicative
capacity in a relatively short span of time; production of healthy and disease free
plants; and its ability to generate propagules around the year (Dhawan and Bhojwani,
1986).
Review of Literature 11
2.2.1 (a) Selection of parent material
Plant material can either be a selected phenotype (a specific tree) or the source
of elite seed (Debergh and Read, 1991; Yasseen et al., 1995 and Ali et al., 2003).
Sometimes the explants from mature trees growing in the field, are avoided because
of the problem of endophytic microorganisms. Instead, the propagates (vegetative or
sexual) from such trees are grown in more hygienic conditions in the green house
(Debergh and Maene,1981; Yasseen et al., 1995; Singh et al., 2002; Ali et al., 2003)
to eliminate the contamination . Hartmann and Kester (1983) and Pierik (1987)
reported that healthy, young and soft explants (actively growing shoots) are generally
more suitable for culture than old woody tissues. They also reported that the
procedure of elimination of contamination was easier in case of small sized explants
but handling was difficult which resulted into poor survival. Various explants of J.
curcas such as leaf segment, petiole, stem, hypocotyl, peduncle and nodal segment,
hypocotyl, epicotyl, and leaf tissue (Sujatha and Mukta, 1996), Sardana et al., (2000),
Rajore and Batra, (2005), Lu et al., (2003); Wei et al., (2004) have been used for
shoot multiplication.
2.2.1 (b) Genotype of parent
Genotype is one of the vital factor in determining the regeneration frequency
and culture ability of plant cell, tissues and organs. Gohil and Pandya (2009) studied
the significant differences among genotypes for most of the characters except primary
branches in various genotypes of J. curcas. The traits like seed yield, plant canopy,
number of leaves, seed oil and kernel oil had higher genetic components than
environmental components. The evaluation of cultivars revealed a good degree of
variation for plant height, stem girth, branches per plant and seed weight. The pattern
of variation exhibited for different characters was found to be different and varied
with age. Such variation among different populations may be due to different
intensities of natural selection acting upon the traits of J. curcas in their natural
habitat (Saikia et al., 2009).
2.2.1 (c) Nature and origin of explants
Both the nature and the origin of explants play a major role in their in vitro
development (evolution) on a certain type of medium and under specific environment
Review of Literature 12
conditions (Cachita, 1991). The buds nearest to the apex and closest to the base of the
stem exhibited the slowest rate of development, but those from the mid-stem region
grew very rapidly. The potential of axillary bud outgrowth, which is related to
position on the main axis, appears to be determined by a balance among several
hormones (Sato and Mori, 2001).
Hard seed coat prevents seed germination by interference with water uptake,
gaseous exchange. Also seed coat supplies inhibitors to the embryo or prevent the
exhibit of the inhibitors (Bewley and Black, 1994). Some germination inhibiting
substances are present in the cotyledons whose affect is reduced when the seeds are
halved (Bewley and Black, 1994). The lowest percentage of seedlings was produced
by halved embryo. This may be because, while dissecting, damage may be caused in
the meristematic region of the embryos. Similar pattern of growth of halved embryos
was also observed in Rubus seed germination (Klee et al., 1985).
2.2.1 (d) Explanting season
In vitro growth of explants, phenolic exuation and degree of contamination
may be influenced by the seasonal conditions at the time of explants collection.
Seasonal qualitative changes in the hormonal content occurs in xylem sap of woody
plants (Alvima et al., 1976). Internal changes in the hormonal status of source plant
may also indicate in vitro behaviour of the explants. Singh et al., (2002) observed that
culture establishment of guava was highest during spring season (April-May),
followed by autumn season (September-October). August and September months
were most ideal for getting maximum regeneration percentage from in vitro shoot tip
and axillary bud culture in papaya cultivars both the explants performed poorly during
the months of December, January and February irrespective of the cultivars used
(Beniwal, 1996).
2.2.1 (e) Explants size
Otoni and Teixiera (1991) studied the influence of the length and position of
nodal segments of Citrus sinensis (L.) on the axillary bud multiplication, they found
that explants size affected the number of axillary shoot development from them; this
number was greater with 0.5 cm nodal segment; however, rooting was best in the
shoots obtained from nodal segments 2.0 and 3.0 cm in length. Pierik (1987) revealed
Review of Literature 13
that it was difficult to induce growth in small explants than in large explants. Kumar
(2003) reported that the bigger size explants (3cm) of guava (Psidium gualala L.)
showed better survival than smaller size explants.
2.2.1 (f) Explants position on the media
Alekhno and Vysotskii (1986) reported that growing shoots of rose in
horizontal position during proliferation almost doubled the axillary branching as
compared with growing shoots in vertical position. Bhatia et al., (2005) studied the
explant orientation on shoot regeneration from cotyledonary explants of tomato.
Cotyledons placed in abaxial (lower surface facing down) orientation consistently
produced better shoot regenerative response and produced greater numbers and taller
shoots compared to those inoculated in adaxial (upper surface facing down)
orientation.
2.2.1 (g) Contamination
The contaminating bacteria and fungi may be endophytic or epiphytic, may be
pathogenic or saprophytic (Debergh and Maene, 1981). Fungus or a bacterium
associated with external plant surfaces (epiphyte) that survive surface sterilization
procedures (Leifert et al., 1989; Wilson and Power, 1989; Gunson and Spencer,
1994). The use of anti-microbial agents (anti-bacterial as well as anti-fungal) to
control contamination is the preferred method (Thurston et al., 1979; Falkiner, 1988;
Wilson and Power, 1989; Kneifel and Leonhardt, 1992). However, their
indiscriminate use may lead to phytotoxicity problems (Phillips et al., 1981; Pollock
et al., 1983).
Murali et al., (2001) recorded that the spraying of mother bushes with
fungicides and antibiotics individually or in the combination, prior to explants
collection reduced the microbial contamination in tea.
Shrivastava and Banerjee (2008) studied that 0.1% mercuric chloride solution
for 2 to 3 minutes were the best surface sterilization method in tissue culture of J.
curcas. Nodal explants (2–3 cm in length) collected from the seven month old donor
plant kept for 3 h in a systemic fungicide, Bavistin (BASF India Ltd) prior to surface
sterilization to remove fungal growth (Shilpa and Batra, 2005). They were surface
sterilized in 0.1% HgCl2 (w/v) for 20–25 min followed by repeated washing (five
Review of Literature 14
times) with sterile distilled water. After sterilization, the explants were trimmed (~1.0
cm) at the base and cultured with the cut surface in contact with the culture medium
(Datta et al., 2007). Gao et al., (2008) studied that seeds were subjected to 70%
ethanol for 30 s, and then to 0.1% mercuric chloride for 8 min to control
contamination. Rashida and Rabia, (2007) studied that the seeds were firstly washed
with commercial detergent (Zip) followed by running tap water. The seeds were then
soaked in autoclaved distilled water for 4 hours to soften the hard seed coat. The seeds
were then surface sterilized with 0.1% mercuric chloride solution for 3 minutes
followed by 4 rinses in sterile distilled water under aseptic conditions.
2.2.1 (h) Explants Exudation
The presence of phenolic compounds causing death of explants has been
another important problem of tissue culture of woody perennials (Compton and
Preece, 1986).When tissues are injured, such as when explants are cut off the stock
plant and during preparation for the in vitro environment, various compounds are
often released that are oxidised and are many times aggravated by growth media
constituents (Seneviratne and Wijesekara, 1996). Polyphenols can be oxidized by air
(Robertson, 1983), peroxidases (Vaugh and Duke, 1984), and turn brown or black.
This may be the reason for the widely reported darkening of both tissue and culture
medium. Tissue blackening occurs due to action of copper-containing oxidase
enzymes: polyphenoloxidases like tyrosinases, which are released or synthesized in
oxidative conditions after tissue wounding and they oxidize o-diphenols released due
to cellular wounding to o-quinones (Scalbert et al., 1988; Marks and Simpson, 1990).
When plants are injured, as in plant preparation, phenolic compounds that are largely
located in the vacuole are mixed with contents of plastids and other organelles, and
then, the dark pigmentation associated with polyphenolic compounds appear. When
phenols become oxidised they form compounds called quinines. These are highly
reactive compounds that polymerize rapidly and form covalent bonds with proteins
(Loomis and Battaile, 1966). Oxidised polyphenolic compounds inhibit enzyme
activity and may result in the decline or lethal browning of the explants.
The onset of tissue browning has been found to be associated with changes in
protein pattern, amino acid content, ethylene production and the occurrence of
Review of Literature 15
saccharose and accumulation of starch. These changes eventually lead to growth
inhibition or death of explants (Lindfors et al., (1990). The various techniques
employed to overcome the harmful effects of browning attempts to avoid the build up
of toxic substances in the medium. These include:
1. Choice of juvenile explants, or new growth flushes during the active growth
period (Amin and Jaiswal, 1987; Bon et al., 1988).
2. Transfer of explants to fresh medium at short intervals (Broome and
Zimmerman, 1978; Lloyd and McCown, 1980; Amin and Jaiswal, 1988;
Murkute et al., 2004).
3. Inclusion of antioxidants in the culture medium, or soaking of explants in
water or solutions containing antioxidants prior to inoculation (Gupta, 1980;
Vieitez and Vieitez, 1980; Ziv and Halevy, 1983; Amin and Jaiswal, 1987;
Shekhawat et al., 1993; Wang et al., 1994; Toth et al., 1994; Dhar and Upreti,
1999).
4. Dry the explant under laminar air flow (Fitchet, 1990).
5. Addition of amino acids like glutamine, arginine and asparagine to the media
(Pierik, 1987).
2.2.1 (i) Media Formulation
Thorpe and Patel (1984) used various mineral salt formulations for in vitro
culture. However, full strength mineral salts are not always optimum and different
formulation says work better at different stages. Modifications with respect to
different constituents like phytohormones, sucrose, agar concentration and other
additives like PVP, AC, coconut milk etc. were usually done in order to ensure a
better in vitro response (Thorpe et al., 1991).
2.2.1 (j) Basal medium
The most widely used culture medium is Murashige and Skoog (1962) (MS
medium), because most plants react to it favourably. It contains all the elements that
have been shown to be essential for plant growth in vitro. It is classified as a high salt
medium in comparison to many other formulations, with high levels of nitrogen,
potassium and some of the micronutrients, particularly boron and manganese (Cohen,
1995). Due to the high salt content, however, this nutrient solution is not necessarily
Review of Literature 16
always optimal for growth and development of plants in vitro (Pierik, 1997). For that
reason, the use of dilute media formulations has generally promoted better formation
of roots, since high concentration of salts may inhibit root growth, even in presence of
auxins in the culture medium (Grattapaglia and Machado, 1998).
2.2.1 (k) Carbon source
Sucrose is cheap, readily available, relatively stable to autoclaving, and readily
assimilated by plants. Other carbohydrates can be also used, such as glucose, maltose
and galactose as well as the sugar-alcohols glycerol and sorbitol (Fowler, 2000). The
carbohydrates added to the culture medium supply energy for the metabolism (Caldas
et al., 1998). The addition of a carbon source in any nutrient medium is essential for
in vitro growth and development of many species, because photosynthesis is
insufficient, due to the growth taking place in conditions unsuitable for photosynthesis
or without photosynthesis (in darkness). Normally, green tissues are not sufficiently
autotrophic under in vitro conditions (Pierik, 1997) and depend on the availability of
carbohydrates in the growing medium.
2.2.1 (l) Agar
Agar, the solidifying agent is the costiest ingredient and other contains
impurities that may affect the growth of the cultured plant tissue. Physical and
chemical analysis revealed large differences in response of culture to brands or types
of agar (Schotten and Pierik, 1998). Agar has traditionally been used as the preferred
gelling agent for tissue culture, and is very widely employed for the preparation of
semi-solid culture media (Torres , 1988). It is a polysaccharide extracted from species
of red algae which are collected from the sea (Torres et al, 1999). Pâques (1991)
pointed out that there is a strong connection between culture medium hardiness,
proliferation ratio and hyperhydration. Normally, an increase in the agar concentration
promotes a reduction in the occurrence of hyperhydration symptoms in plants. Filter
paper was effective in papaya seed germination may be because it could provide
appropriate moisture necessary for the seeds to germinate as observed in seed
germination of other crops. Cotton holds excessive moisture, which may result in
fungal contamination. Proper humidity can be maintained with filter paper as media
can be replenished as and when needed. Sterilized soil used for in vitro germination
Review of Literature 17
was found to be less suitable than non autoclaved soil used in ex vitro germination.
An alternative to agar is the use of a gelling agent named gelrite. Gelrite is a gellan
gum – a hetero-polysaccharide produced by the bacterium Pseudomonas elodea
(Kang et al., 1982). Gelrite is an attractive alternative to agar for plant tissue culture
because its cost per liter of medium is lower, and it produces a clear gel which
facilitates the proper observation of cultures and their possible contamination
(George, 1993). Williams and Taji (1987) found that several Australian woody plants
survived best on a medium gelled with gelrite rather than agar. The gellan type of
gelling agents such as Gelrite and Phytagel (Sigma) are known to induce
hyperhydricity (Podwyszynska and Olszeweski, 1995). Though responsible for higher
water availability and hyperhydricity in tissue cultures, phytagel was found to be
inferior to agar as gelling agent in seed germination. The positive effect of agar on
seed germination may be due to impurities in agar that acted as medium supplements
(Podwysznska and Olszeweski, 1995).
2.2.1 (m) Phytohormones
Growth regulators or phytohormones are not nutrients, but they influence
growth and development .They are generally produced naturally in plants. Carsells et
al., (1982) reported that in vitro development and organogenetic potential of various
physiological states are influenced by the level of endogenous growth regulators.
Growth and morphogenesis in vitro are regulated by the interaction and balance
between the growth regulators supplied in the medium, and the growth substances
produced endogenously (George, 1993). A balance between auxin and cytokinin is
most often required for the formation of adventitious shoots and roots. High levels of
auxin relative to cytokinin stimulated the formation of roots, whereas high levels of
cytokinin relative to auxin led to the formation of shoots (Taiz & Zeiger, 1991).
Auxin, known to be involved in cell enlargement, was long thought to be the
controlling factor in the rooting process. Two types of evidence support this
reasoning: (a) the increased content of endogenous auxin in the base of cutting during
rooting induction (Blachova, 1969) and (b) the rooting response of many plants to
exogenous auxin. Cultures, however, usually do not manufacture sufficient quantities
of growth regulators, so they must be added selectively to culture media. Gibberellins
Review of Literature 18
are a group of compounds that is not necessarily used in the in vitro culture of higher
plants. In some species, these growth regulators are required to enhance and in others
to inhibit growth (Razdan, 1993).
Thorpe (1983) reported that plant growth regulators (PGR) are important
factors, which can selectively influence the genes triggering differentiation of cells in
culture BA and IBA were shown to be effective growth regulators for the induction of
callus and shoot regeneration from various explants of J. curcas, and their optimal
concentrations ranged from 0.1 mgL-1
to 1 mgL-1
(Sujatha and Mukta, 1996; Lian et
al., 2002; Wei et al., 2004). The IAA is a natural auxin, whereas 2, 4-D and NAA are
synthetically produced and have similar effect in comparison to natural-occurring
auxins. Benelli et al., (2001) and Tanimoto (2005) have proved that IBA is the most
effective auxin in olive rhizogenesis as compared to NAA. The reason for these
differences in root inducing ability may be the slow and continuous release of IAA
from IBA (Krieken et al., 1993; Liu et al., 1998) and release of IBA through
hydrolysis of conjugates (Epstein & Muller, 1993). These IBA conjugates were
reported to be superior to free IBA in serving as an auxin source during later stages of
rooting (Staswick et al., 2005). Rooting was effectively achieved on MS
supplemented with IAA and IBA from shoots of J. curcas (Kalimuthu et al., 2007,
Thepsamran et al., 2007).
2.2.1 (n) Other additives
Glutamine was reported to stimulate growth and somatic embryos formation in
date palm (Hameed, 2001; Jasim and Saad, 2001). The effectiveness of glutamine to
promote shoots regeneration and multiplication of date palm in vitro culture. This
might be due to the rapid uptake of reduced nitrogen which provided by this amino
acid (Abo El-Nil, 1986).
Glutamine and glutamic acid are directly involved in the assimilation of NH4+.
A direct supply of these amino acids should therefore enhance the utilization of both
nitrate and ammonium nitrogen and its conversion into amino acids (George, 1993).
The addition of glutamine in date palm tissue culture media increased callus quality
and somatic embryos formation (Jasim and Saad, 2001), shoots vegetative
multiplication (Hameed, 2001) and adventitious bud multiplication (Khierallah,
Review of Literature 19
2007). Recently, use of additives such as arginine in addition to IBA and BA into the
culture medium was reported to result in 100% survival of tissue-cultured J. curcas
plants (Shrivastava and Banerjee, 2009).
2.2.1 (o) Culture environment
It is the interaction between plant material, culture media, type of culture
vessel and the external environment of the culture room and has much influence on a
tissue culture system (Debergh, 1988). Bonga (1987) reported that many factors of
culture environment influence in vitro growth and differentiation of plant tissue
cultures.
2.2.1 (o1) pH
The pH affects nutrient uptake as well as enzymatic and hormonal activities in
plants (Bhatia et al., 2005). The optimal pH level regulates the cytoplasmic activity
that affects cell division and the growth of shoots and it does not interrupt the function
of the cell membrane and the buffered pH of the cytoplasm (Brown et al., 1979). The
changes in external pH have a small transient effect on cytoplasmic pH but the cells
are readily readjusted towards their original pH, thus the effect of external pH on
cytoplasm is not long lasting. Exposure of cells to extreme low pH leads to
conversion of inorganic phosphate into organic phosphate at the extracellular region.
This is also accompanied by a reduction in ATPs which leads to reduced plant growth
(Mimura et al., 2000). The detrimental effects of adverse pH are generally related to
an imbalance in nutrient uptake rather than to direct cell damage. The pH also
influences the status of the solidifying agent in a medium: a pH higher than 6
produces a very hard medium and a pH lower than 5 does not sufficiently solidify the
medium (Bhatia and Ashwah, 2005).
2.2.1 (o2) Humidity and gaseous atmosphere
Humidity in the vessel and osmotic potential of the medium influence the
water relations of plantlets in vitro, and thus effects the growth, development and
photosynthesis in different ways (Brown et al., 1979; Ziv and Halvey 1983).The
utilization of tightly closed vessels that reduce the gas exchange may affect negatively
the normal growth and development of plants during cultivation in vitro (Campostrini
& Otoni, 1996). Several studies have shown the advantages of using closures with
Review of Literature 20
filters or vented vessels, which allow gas exchange, increasing the photosynthetic
capacity, the multiplication rate, and the survival of plants after transfer to ex vitro
conditions (Chuo-Chun et al., 1998; Murphy et al., 1998; Zobayed et al., 2000;
Gribaudo et al., 2003; Lucchesini and Mensuali, 2004; Park et al., 2004). Since the
gas exchange between outside air and inner air can influence the microenvironment of
culture vessel, it is necessary to measure the air exchange rate for various vessels.
Water vapour was used as the tracer gas, and the change of absolute humidity
inside the vessel was calculated continuously by the measured values of a relative
humidity sensing element. The outside environment was maintained at constant
humidity level by a saturated salt solution. Magdalita et al. (1997) reported that the
accumulation of ethylene in papaya cultures tends to cause an increase in senescence:
3.5-fold higher when the ethylene concentration is 50 ppm as compared to controls, in
nodal culture. Ethylene accumulation can be reduced by adding a loose cap of
aluminum foil and using large culture vessels to increased aeration.
2.2.1 (o3) Light
Light is an important environmental factor that controls plant growth and
development, since it is related to photosynthesis, phototropism and morphogenesis
(Salisbury and Ross, 1994; Read and Preece, 2003). The three features of light, which
influence in vitro growth, are wavelength, flux density and the duration of light
exposure or photoperiod (George, 1993). Several studies showed that light enhanced
root formation and shoot growth (Cabaleiro and Economou, 1992; Cui et al., 2000;
Lian et al., 2002; Kumar., 2003), whereas in others darkness favoured root formation
(Hammerschlag, 1982). The reduced rooting in presence of light is due to the
degradation of the endogenous IAA (George, 1993). In cotton, Tort (1996) concluded
that illumination significantly increased the germination percentage. Compared to
darkened conditions 16 hours photoperiod was found to increase germination percent
and the root growth. Positive effect of light governing seed germination was observed
earlier in other crops (Van der Sman et al., 1992). Light sensitivity of seed was
suggested as having some relations to seed germination in their natural habitat (Mayer
and Polijakoff, 1979).
Review of Literature 21
Sardana et al. (2000) reported the influence of light intensity on somatic
embryogenesis in J. curcas .Maintenance of leaf segments of J. curcas in continous
light elicted only a low response (20%) whereas complete darkness induced 40 % and
a photoperiod of 16 hours was found optimal.
2.2.1 (o4) Temperature
Temperature controls the relatively humidity in the culture vessel, soil has an
indirect effect on vitrification. Temperature influence on various physiological
processes, such as respiration and photosynthesis, is well known and it is not
surprising that it profoundly influences plant tissue culture and micropropagation. The
most common culture temperature range has been between 20°C and 27°C, but
optimal temperature vary widely, depending on genotype (Altman, 2000; Read and
Preece, 2003). During acclimatization, the temperature of root zone is important to
enhance root growth, medium should be warmer than the air for good root activity.
Temperature was found to strongly influence the papaya seed germination.
None of the cultivars germinated at a temperature of 150C and above 40˚C.
Germination percentage was low at 20˚ C and increased with increasing temperature
to a maximum of about 80% at 30˚C. Above 30
˚C, in all the varieties percentage
germination decreased with temperature and was very low at 40˚C (Yahiro and
Yoshitaka ,1982).
Direct embryogenesis observed in J. curcas has a great potential for its
application in mass propagation. Concentration of TDZ in the medium significantly
influenced the response of shoot bud induction irrespective of genotype. The
percentage of induction of shoot buds and the number of induced shoot buds per
explants were directly proportional to the concentration of TDZ . Datta et al., (2007)
investigated that axillary shoot bud proliferation was best initiated on Murashige and
Skoog’s (MS) basal medium supplemented with 22.2 μM N6-benzyladenine (BA) and
55.6 μM adenine sulphate and produced 6.2 ± 0.56 shoots per nodal explants with 2.0
± 0.18 cm average length after 4–6 weeks. The rate of shoot multiplication was
significantly enhanced after transfer to MS basal medium supplemented with 2.3 μM
6-furfuryl amino purine (Kn), 0.5 μM indole-3-butyric acid (IBA) and 27.8 μM
adenine sulphate for 4 weeks. Both shoot number (30.8 ± 5.48) and average shoot
Review of Literature 22
length (4.8 ± 0.43 cm) were found to increase significantly. About 52% of root
induction occurred in MS basal medium supplemented with 1.0 μM IBA in 2–3
weeks. Further elongation of roots with average length of 8.7 ± 1.35 cm was obtained
in unsupplemented MS basal medium for 2–3 weeks. Kalimuthu et al. (2007)
reported that nodal explants of biodiesel plant J. curcas micropropagated on MS
supplemented with BAP (1.5 mgL-1
), Kn (0.5 mgL-1
) and IAA (0.1 mgL-1
).
Thepsamran et al. (2007) studied an appropriate in vitro multiple shoot induction
medium for establishment of J. curcas (L.) from various explants. Shoot regeneration
from apical shoots, nodes, axillary bud-derived shoots, petioles and leaf explants were
assessed on Murashige and Skoog (MS) medium supplemented with different
concentrations of N6-benzyladenine (BA) alone, or in combination with indole-3-
butyric acid (IBA). MS medium with 2.22-4.44 µM BA was effective for apical shoot
culture, while 4.44 µM BA was suitable for node culture. MS medium with 2.22 µM
BA and 0.049 µM IBA provided the best shoot proliferation from axillary bud-
derived shoots.
Sardana et al., (2000) developed an expeditious method to induce somatic
embryogenesis in J. curcas, a 2-stage method was established.Globular embryos
differentiated on media containing MS +B5 vitamins+ IAA (mgL-1
) +BAP (mgL-1
)
which matured by following normal embryogeneic stages on the medium which
contained MS + IAA (1 mgL-1
) + GA3 (3 mgL-1
).To promote germination, MS
medium was supplemented with 3% sucrose and the regenerated plantlets were
transferred to pots . Callus induction from hypocotyls, petiole and leaf explants of J.
curcas was evident within two weeks of incubation when auxins were used singly
except of IAA. Usage of cytokinins not only slowed down the response but was also
coupled with necrosis (Sujatha and Mukta, 1996). Callus proliferation is important in
all environments, planting and proliferation, as reported by Weida et al., (2003),
during the regeneration of plantlets from explants of hypocotyls and petioles of
J. curcas leaves in a medium supplemented with BA and IBA. Callus induced from
hypocotyls, petioles and leaf explants of J. curcas on auxin supplemented medium
exhibited shoot bud formation upon transfer to a medium containing IBA and Kn (4.9
µM). Shoot induction was evident irrespective of the explants source, growth
Review of Literature 23
regulator type or concentration (Sujatha and Mukta, 1996). The explants of hypocotyl,
leaf blade and petiole from J. curcas were cultured on Murashige-Skoog (MS)
medium with indole-3-butyric acid (IBA) and N6-benzyladenine (BA) for induction of
callus (Lu et al., 2003; Wei et al., 2004). Regenerated shoots could be rooted on
growth regulator free MS medium and could be transplanted in soil after simply
hardening for several days (Lu et al., 2003). Regenerated plants with well developed
shoots and roots were successfully transferred to greenhouse, and the survival rate
was 81.6% (Lu et al., 2003).Thepsamran et al. (2007) studied that for petiole
segments from leaves at the second, third and fourth nodes, the most effective growth
regulator combination for callus induction during the first culture cycle, and
subsequent shoot formation during the second culture cycle, was MS medium
supplemented with 4.44 µM BA and 2.46 µM IBA. A combination of 8.88 µM BA
and 4.90 µM IBA was suitable for callus induction and shoot formation from leaf
segments at the second and third nodes of J. curcas branches.
Somatic embryogenesis, a powerful tool of plant biotechnology for faster and
quality plant production has been successfully applied to regenerate plants in J.
curcas for the first time. Embryogenic calli were obtained from leaf explants on MS
basal medium supplemented with only 9.3 lM Kn. Induction of globular somatic
embryos from 58% of the cultures was achieved on MS medium with different
concentrations of 2.3–4.6 lM Kn and 0.5–4.9 lM IBA; 2.3 lM Kn and 1.0 lM IBA
proved to be the most effective combination for somatic embryo induction in J.
curcas. Addition of 13.6 lM adenine sulphate stimulated the process of development
of somatic embryos. Mature somatic embryos were converted to plantlets on half
strength MS basal medium with 90% survival rate in the field condition (Nataraja et
al., 1973; Jha et al., 2007). Soomro and Memon (2007) studied that callus cultures
were initiated from leaf and hypocotyl explants isolated from 4 days old seedling of J.
curcas (L.), on Murashige and Skoog (1962) basal medium supplemented with
different growth regulator formulations including 2,4-D, BA, GA3, and coconut milk.
Excellent growth of callus was obtained in medium supplemented with 0.5 mgL-1
2, 4-D alone and with 2% v/v coconut milk in hypocotyl explants, Callus produced
from hypocotyl explants grew faster during 7 to 30 days of culture then stabilized at a
Review of Literature 24
low growth rate. Calli cultured on this medium showed 8 fold increases in fresh
weight by the fourth week of incubation. Callus was soft, friable, globular, lush green
in colour. Hypocotyl explant and 0.5 mgL-1
2, 4-D proved to be most effective in
inducing of callus on a large scale in short period of time. The friable green callus was
then used for establishment of homogeneous and chlorophyllous suspension culture.
Maximum growth of suspension culture was achieved in medium supplemented with
0.5 mgL-1
2, 4-D, with initial inoculum cell density of 1%. The growth rates of cells
were initially slow but as the cultures proceeded, the growth increased significantly
and accumulated a great amount of fresh weight (5-fold) over a period of 21 days then
the growth of cells was stable for 30 days. Wei et al., (2004) regenerated J. curcas
from epicotyls explants taken from one month old tube plantlets grown from extracted
embryo of seeds regenerated on MS medium supplemented with IBA (0.1 mgL-1
) and
BA (0.2-0.7 mgL-1
) .They found combination of IBA (0.1 mgL-1
) and BA (0.5 mgL-1
)
was the best for the induction of adventitious buds and shoot formation. They
succeeded in inducing roots on same medium without hormone. Regenerated plants
with well developed roots and shoots were successfully transferred to pots. Lin et al.,
(1997) successfully induced adventitious buds from hypocotyl explants and the
induction frequency was 21% while Wei et al. (2004) obtained higher induction
frequency of 38% from epicotyls explants. Also the other explants like leaf, petiole,
cotyledons and hypocotyl have inductive frequencies ranging from 44-56% (Lu et al.,
2003). De Lange et al., (1978) reported internode and petiole explants of Euphorbia
pulcherrima to be suitable for in vitro propagation.
Deore and Johnson
(2008) developed a simple, high-frequency and
reproducible protocol for induction of adventitious shoot buds and plant regeneration
from leaf-disc cultures of J. curcas (L.) supplemented with thidiazuron (TDZ)
(2.27 μM), N6 Benzyladenine (BA) (2.22 μM) and indole-3-butyric acid (IBA)
(0.49 μM). The presence of TDZ in the induction medium has greater influence on the
induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted
callus induction rather than shoot buds. Induced shoot buds were multiplied and
elongated into shoots following transfer to the MS medium supplemented with BA
(4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and
Review of Literature 25
gibberellic acid (GA3) (0.72 μM). Well-developed shoots were rooted on MS medium
supplemented with IBA (0.5 μM) after 30 days. Sujatha and Mukta (1996) recorded
direct adventitious shoot bud induction highest on MS medium supplemented with
2.22 µM BA and 4.9µM IBA.Reduced IBA concentration (0.49 µM ) proved effective
in callus mediated regeneration from hypocotyls and leaf explants, the petioles
required lower concentrations of two growth regulators (0.44µM BA and 0.49 µM
IBA).
The successful acclimatization of micropropagated plants and their
subsequent transfer to the field is a crucial step for commercial exploitation of in vitro
technology. Preece and Sutter (1991) have reviewed acclimatization of
micropropagated plants in the greenhouse and in the open field. Kalimuthu et al.,
(2007) reported that hardening experiment in J. curcas showed the commercial
medium containing a mixture of decomposed coir waste, perlite and organic compost
in the ratio of 1:1:1 by volume was most effective, 80% plantlets survived. The
substrate, in which the plants develop secondary roots, and the container used are of
fundamental importance in the acclimatization phase. The substrate must allow the
formation of roots in this phase, but also depends on the type of container used. For
acclimatization, trays are used and the reduced height of the cells determines special
patterns of drainage, with greater retention of water. These containers are known as
plugs and can be defined as the smallest possible volume for the production of an
individualized seedling. The smaller the plug, the higher is the vulnerability to
humidity changes, nutrient deficiency, oxygenation, pH and soluble salts (Styer and
Koranski, 1997). The cell height in the trays influences the drainage due to the effect
of the gravity force, with larger amount of water accumulated in the bottom of the
plug. This problem can be solved with the use of substrates with porosity above 85%.
Styer (1997) mentioned two important characteristics that should be taken into
account when choosing a substrate, namely water holding capacity and aeration space
of the substrate. The water holding capacity determines the irrigation frequency and is
related to the presence of capillary pores.The opposite is the aeration space, which is
determined by the distribution of no capillary pores (macropores).
Review of Literature 26
2.2.1 (p) In vitro grafting
Shoot-tip grafting (STG) in vitro, which was studied by Murashige et al
(1972) and described in details by Navarro et al., (1975), is the most effective
technique for elimination of all major virus and virus-like pathogens, including those
not eliminated by thermotherapy. Plants obtained by STG are true-to type and they do
not have juvenile characters. Thus, these plants could be used for budwood production
after they are indexed (Navarro, 1988). In order to shorten the growing period of
plants for indexing stage, some modified procedures and methods, i.e. micrografting
technique, have been developed (De Lange, 1978). Navarro (1988) developed a
method of shoot-tip grafting in vitro to obtain virus-free peach plants. It consists of
aseptically grafting a 0.5 – 1.0 mm long shoot tip excised from a diseased plant on a
10-to-14-day-old Nemaguard seedling grown in vitro. Shoot tips were composed of
the apical meristem and three to four leaf primordia. Between 45 and 70% of
successful grafts were obtained and 60 to 70 % of the micrografted plants survived
transplanting to soil.
Oguz et al., (2009) reported that the scions of apple cultivars were whip-
grafted onto rootstocks to observe graft union development. Graft samples were taken
on 15th
and 30th
day and every 30 days thereafter for a year. Samples of 5 graft unions
from each scion/rootstock combinations were fixed in ethanol (70%). Successful graft
unions were observed in 90 days samples in all combinations and no evidences for
tissue incompatibility found in this study. Grafting success (union of grafts) was
significantly dependent on the method of grafting. The highest percentage (65%) of
successful grafts was obtained with the homoplastic apex graft (shoot-tip), with apical
bud scion length greater than 6 mm. However, the success of heteroplastic side bud
apices (wedge) grafts was only 16%. The homoplastic micrografting method in vitro
can be a useful technique in the improvement, rejuvenation and freeing from viruses
for fruit crops. In vitro germinated sour cherry seedlings (Prunus cerasus ‘Albaloo’)
which emerged one month after inoculation were decapitated and used as rootstocks.
One month old in vitro-cultured meristematic apices with length of 5–15 mm were
used as microscions. The highest amount of successful grafts was obtained with the
homoplastic apex graft (shoot-tip), when compared with wedge grafts and
Review of Literature 27
heteroplastic grafts. The graft union, as measured by graft compatibility, was
satisfactory, although, it formed very slowly during the first month (Amiri et al.,
2006). Wu et al. (2007) developed a successful shoot-tip micrografting technique
using in vitro-germinated P. cynaroides seedlings as rootstocks and axenic
microshoots established from pot plants as microscions. Thirty day old seedlings,
germinated on growth-regulator-free, half-strength Murashige and Skoog medium,
were decapitated and a vertical incision made from the top end. The bottom ends of
microshoots established on modified Murashige and Skoog medium were cut into a
wedge (‘V’) shape, and placed into the incision. In vitro grafting of cotton shoots to
seedling rootstock proved to be a simple and reliable method allowing 90–100%
recovery of non-rooting shoots from culture. Success of any given graft was directly
related to scion size (0.8–1.0 cm) and age (14–35 days) of the seedling rootstock
(Jinhua and Jean, 1999).
Horizontal and wedge grafts utilized with micropropagated prickly pear cactus
(Opuntia sp.) to determine the best method for in vitro micrografts. Different
genotypes were used as rootstocks and combined with O. ficus-indica to produce
homo and heterografts. The easiest and most successful method for micrografting
prickly pear cactus was the horizontal graft. Within 28 days vascular connections
occurred within the callus bridge between rootstocks and scions in all genetic
combinations. Homografts (grafts between same species) grew significantly faster
than the heterografts (grafts between different species) after 90 days of ex vitro
transfer (Estrada et al., 2002). Micrografting has several unique uses including:
production of disease-free plants by grafting small meristem tips (Zilka et al., 2002),
virus indexing by micrografting to susceptible understocks (Zimmerman, 1993), early
detection of grafting incompatibility relationships (Jonard, 1986), propagation of
novel plants created in tissue cultures that are difficult-to-root (Barros et al., 2005)
and small micrografted trees are a convenient way to exchange germplasm between
countries (Navarro et al., 1975).
2.2.1 (p) Abiotic Stress
Soil salinity is a prevalent abiotic stress for plants. Growth inhibition is a
common response to salinity and plant growth is one of the most important
Review of Literature 28
agricultural indices of salt stress tolerance as indicated by different studies (Parida and
Das, 2005). In order to define salt stress tolerance or sensitivity of seedlings, the
effect of NaCl treatment on fresh weights of the cotyledons, hypocotyls and radicles
were tested. The fresh weight of cotyledons and radicles showed no significant
changes at NaCl concentration of 50 mmol, but they decreased progressively with
increasing NaCl concentration. The fresh weights of hypocotyls decreased gradually
up to 150 mmol NaCl concentration then increased at NaCl concentration of 200
mmol. The cotyledon area and height of seedlings were the most sensitive to salt
stress, especially under higher NaCl concentrations, which led to below medium of
seedlings more sensitive to salt stress than above medium. 20% of the world’s
cultivated and nearly half of the world’s irrigated lands are affected by salinity (Zhu,
2001). Moreover, salt stress has become an ever increasing threat to food production,
irrigation being a major problem of agricultural fields due to gradual salinization
(Rabie and Almadini, 2005). Suppression of growth occurs in all plants but their
tolerance level and rates of growth reduction at sub lethal concentrations of salt vary
widely among different plant species. To achieve salt tolerance, three interconnected
aspects of plant activities are important. First, damage must be prevented or
alleviated. Second, homeostatic conditions must be re-established in the new stressful
environment. Third, growth must resume albeit at a reduced rate (Zhu, 2001).
Gao, et al., (2008) investigated the effects of increasing NaCl concentrations
on biomass, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and
phenylalanine ammonia-lyase (PAL) in J. curcas (L.) seedlings . The fresh weights of
cotyledons and radicles with increasing NaCl concentrations decreased progressively,
and the fresh weight of hypocotyls reached the lowest level at NaCl concentration of
150 mmol and then increased. SOD activity in the cotyledons, hypocotyls and radicles
increased gradually up to NaCl concentrations of 150 mmol, 200 mmol and 150
mmol, respectively. The highest POD activities in the cotyledons, hypocotyls and
radicles were observed at NaCl concentrations of 150 mmol, 200 mmol and 150
mmol, respectively. CAT activity in the cotyledons, hypocotyls and radicles enhanced
gradually up to 100, 200 and 150 mmol NaCl concentrations, respectively. Increased
PAL activity in the hypocotyls and radicles was linearly and positively correlated with
Review of Literature 29
increasing NaCl concentrations, but the peak activity in the cotyledons was observed
at NaCl concentration of 150 mmol.
2.2.2 Seed
Using generative propagation, direct seed sowing is recommended at the
beginning of the rainy season, after the first rain when soil is wet, because it helps to
develop a healthy taproot system (Gour, 2006). Seedlings can be pre-cultivated in
polythene bags or tubes or in seed beds under nursery conditions. The use of plastic
bags or tubes is observed to induce root node formation and spin growth (Soares et
al., 2007). In the nursery, seeds should be sown three months before the rainy season
in a soil with a high concentration of organic material (sandy loam soil – compost
ratio 1:1 (Kaushik et al, 2006); in case of more heavy soils, sand is added: sand–soil
compost ratio 1:1:2 (Gour, 2006); sand – soil – farm yard manure ratio 1:1:1 (Singh
et al., 2006) and should be well watered (Henning, 2000). Pre-soaked seeds (24 hours
in cold water) germinate in 7-8 days in hot humid environment whereas the process
continues for 10-15 days (Gour, 2006). Kaushik et al., (2006) studied the effect of
maturity stage of seed on germination, root length, shoot length and vigour index in J.
curcas seedlings. He found that yellow fruit (mature seed ) harvested at 57 DAA gave
higher germination percentage, root length, shoot length and vigour index and also
studied the effect of seed size on J. curcas seedlings growth after 90 days of sowing.
He found that large seed size in the rooting media containing FYM resulted in the
production of better seedling of J. curcas.
Islam et al., (2009) observed that highest germination percentage (95.85%)
when seeds were kept under stone sand and moistened once with the water and 100%
germination when seed was placed on filter paper in the petri dish and moistened once
with the water. Saikia et al., (2009) investigated that growth traits, viz. height, stem
girth, 100 seed weight and field survival have significant inter correlation with each
other. It was found that heavier seeds have better seedling growth in the field (Aslan,
1975). The correlation suggests that following the complection of germination,
seedlings allocate much of their energies for root and shoot development. The inter-
correlation found among seed weight and seedling characters in J. curcas is consistent
with that of earlier studies (Palmberg, 1975; Iktueren, 1977; Isik, 1986; Ginwal, et al.,
Review of Literature 30
2004). Seedling shoot-length and dry matter yield were significantly affected by seed
weight. Seedlings grown from the heaviest seeds were 51% taller and 91% heavier
than those from the lightest ones .The improved seed and seedling quality, as
associated with greater seed weight, is attributed to better membrane integrity and
increased availability of energy in the endosperm (Zaidman, et al., 2010). Meng Ye
et al., (2009) studied that in direct seeding, soil moisture needs to be high. The
recommended number of seeds is 4–7 per hole, with 3–5 cm soil covering. The
appropriate season for seedling planting is in June or July.
2.2.3 Cuttings
J. curcas is easily propagated by vegetative (direct planting of cuttings)
methods. For quick establishment of living fences and plantations for erosion control,
direct planting of cuttings is considered easier (Heller, 1996), although J. curcas
plants propagated from cuttings do not develop a taproot.The plants only develop thin
roots unable to grow deep in the soil, which makes the plants more susceptible to
uprooting by wind (Soares et al., 2007). Cuttings of 25-30 cm length from one-year-
old branches (Gour, 2006) or longer cuttings up to 120 cm (Henning, 2000) are
among the options. Kaushik & Kumar (2006) report that the survival percentage
depends on the origin of the source material (top, middle or base of the branch) and
the length and diameter combination of the cutting (Kaushik and Kumar, 2006) .Their
study showed a survival percentage of 42% when the top of the branches were used as
cuttings, while cuttings from the middle (72%) and base (88%) showed significantly
better survival results. According to Hartmann and Kester (1983), the following two
factors are generally responsible for sprouting: the age of the plant from which
cuttings are taken and the position of the cutting within the plant. Cuttings can be
generated from one or two year old twigs with 15–20 cm length. The proper time for
raising cuttings is from the last ten days of August to the first half days of September.
Cuttings may be covered with an arched roof made of plastic film, in which the
temperature should not exceed 30°C. Rooting begins after 30–45 days of planting, and
the generation rates from cuttings range from 50 to 80%. Narin and Stienswat (1983)
showed that treating cuttings with IBA (indole-3-butyric acid) hormone did not
promote root formation. The rooting of stem cuttings is influenced more by rooting
Review of Literature 31
media; good aeration and drainage proved profitable. Kochhar et al., (2005) studied
that two Jatropha species exhibit differential response to external auxin application,
which is dependent on the endogenous auxin status of the cuttings. A more interesting
observation is that shoots are formed much earlier in Jatropha species than roots.
Shoots thus formed earlier due to reserve carbohydrates, start producing auxins which
moves downward, thereby accumulating in the lower portion of the cuttings. When
the concentration reaches a threshold value, endogenous auxins at the extreme basal
end start getting metabolized and signal the process of root initiation. Nanda and
Kochhar (1987), while studying the rooting behaviour of 100 Indian forest tree
species, had shown that a balance of auxin and carbohydrates determines the ability of
cuttings to root. Kathiravan et al., (2009) studied the effect of cuttings length and
thickness on plant height and no. of branches in J. curcas. Macropropagation of stem
cuttings with 40 cm length and 2.5 to 3.0 cm thickness is successful in generating
higher survival with biomass productivity. Kaushik et al., (2006) investigated the
effect of date of planting and growth hormones on rooting and sprouting of J. curcas
cuttings. The cuttings planted on 1st and 15
th march showed better results in terms of
rooting and root length. Application of IBA at the rate of 50 and 100 ppm was more
effective as compared to control and NAA at all the levels of concentrations. First
fortnight of March was found optimum for planting J. curcas cuttings in the nursery.
Saikia et al., (2009) observed the pattern of root formation of J. curcas
cuttings of different diameters (1, 2 and 3 cm) and lengths (5 and 7 cm) in the
polythene bags. Thicker cuttings formed more roots than the thinner ones. Cuttings of
30 cm length developed more roots and their survival rate was higher than cuttings of
15 cm length. The following two factors are generally responsible for rooting: age of
the plant from which cuttings are taken and position of the cutting within the plant
(Hartmann and Kester, 1983) .
2.2.4 Grafting
Lim et al., (1996) studied that grafted trees yields earlier than those developed
from seedlings.The scion should be taken from terminals at the stage of a new flush
with buds which are swollen but have not opened. For the forkert bud graft a bud
together with a strip of underlying bark and cambium about 20 mm long and 5mm
Review of Literature 32
wide is cut from the mother plant.For rootstock two parallel cuts 5mm apart and
20mm long are made down the length of the stem a few centimetres above the
ground.The cuts go into the cambium.The scion is fitted into the hollow, the flap is
cut off leaving a small tag to hold the scion in place and the graft is bound with
grafting tape.About two weeks after the scion starts flushing the rootstock above the
graft should be cut off so that scion bud becomes the only shoot on the plant. Joolka et
al., (2001) observed that high bud sprouting were recorded in tongue grafting in pecan
performed on 3rd
March followed by 15 Feb. While standerdizing of top working
technique in wild pomegranate trees, Kar et al., (1989) found side veneer grafting as
the best which gave 100% success when done on first and 15th
July. Dhillion et al.,
(2009) reported that cleft grafting was more successful during monsoon and spring
seasons for union of J. curcas as scion and J. gossypifolia as rootstock in terms of
initial sprouting of scion at two weeks after grafting .Such findings were also reported
in Madhuca latifolia, Azadirachta indica and Prosopis species (Jagatram et al., 2002;
Dhillion and Hooda, 2005; Jalil and Kashyap 2006). It was also found that top cleft
grafting was strong and stable than other grafting methods including side cleft
grafting. Higher grafting success rate during monsoon season in comparison to spring
season seems to be due to high atmospheric humidity and increased sap content in
stocks and scions besides minimum transpiration losses. The shortening of time
period, easy union of genotypes and early flowering induction are the main character
features of the grafting.
2.3 Cost of production
The major application of plant tissue culture lies in the production of true-to-
type high quality planting material that can be multiplied under aseptic conditions on
a year round basis any where irrespective of season and weather . Micropropagation is
a capital intensive technology involving energy and labour. This problem has been
addressed by inventing reliable cost effective tissue culture without compromising on
quality of plants. Cost of chemical inputs, media, energy, labour and capital counts on
production costs. The cost of medium preparation can account for 30-35% of the
micropropagated plant production. Therefore, low cost alternative are needed to
Review of Literature 33
reduce cost of production of tissue cultured plants (George, 1993; Anonymous, 2004).
Low cost technology means an advanced generation technology in which cost
reduction is achieved by improving process efficiency and better utilization of
resources (Savangikar, 2002). The composition of the culture media used for shoot
proliferation and rooting has tremendous on production costs. Sugar and Agar add
significantly in the media cost. Use of household sugar liquid medium helps in
reducing cost of production to certain extend,without compromising quality of
plants.Water is the main compound of all tissue culture media. Distilled water
produced through electrical distillation is very expensive and use of alternative water
sources can be to lower the cost of medium (Prakash et al., 2002). Raghu and Martin,
(2007) observed that liquid medium and low cost substitute such as sugar and tap
water did not adversely affect the proper growth of the plant. The self sustainable
project for in vitro clonal propagation of papaya was suggested by Beniwal (1996)
with an estimated cost of Rs.5.86 per transplant.