REVIEW OF LITERATURE - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/7958/8/08... · 2018. 7. 9. · plants and though his attempt was unsuccessful, ... totipotency that refers

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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


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).


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


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


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


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


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


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


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


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,

http://s.wanfangdata.com.cn/paper.aspx?f=detail&n=10&q=%e4%bd%9c%e8%80%85+%3a+%22Thepsamran%2c+N%22++DBID%3aNSTL_QK


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


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).


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


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



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



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


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).


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


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


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


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.,

http://scialert.net/asci/author.php?author=A.K.M.A.&last=%20Islam


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


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


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


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.

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