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5. DISCUSSION:

Age of donor tree (maturity level) strongly influenced the rooting of stem

cuttings as well as in vitro propagation (Amri et al. 2010; Liu and Pijut 2008; Husen

and Pal 2007; Bhardwaj and Mishra 2005; Stenvall et al. 2004; Dumas and Monteuuis

1995). In A. excelsa and T. undulata also rooting is easy from seedling/juvenile

explants as compare to mature trees (Varshney and Anis 2011; Sharma 1999;

Nandwani et al. 1995). Unfortunately in trees, important genetic traits (e.g., wood

quality, tree shape, fruit quality) are identifiable only at maturity age.

Micropropagation protocols through seedlings have limited advantage in case of tree

species (Gupta and Durzan 1987). Therefore, in present research work all efforts

were focused with mature trees to develop clonal techniques of Tecomella undulata

and Ailanthus excelsa.

Presently investigated two species posses different kind of problems during

macro and micropropagation stages and both are difficult to root species (Sharma

1999; Bhansali 1993). Some important achievements of present research work are

highlighted below before discussing the results in details.

A. Micropropagation in T. undulata:

1. In vitro shoot cultures can be established throughout the year but highest

percentage of bud break (75 %) was observed in January-February. Bud break

percentage was not influenced by NAA and BA added in MS or B5 medium.

But MS medium was better than B5 medium. However, NAA (0.1 mg/l) and

BA (2 mg/l) enhanced shoot length and multiplication, respectively.

2. MS + 1 mg/l BA + 3 % sucrose was found best for shoot multiplication with

high frequency of rootable shoots (above 2.5 cm). Shoot can be multiplied

upto 4 years but 3rd year onwards shoot multiplication declined gradually.

3. A combination of IBA + NAA (100 mg/l) each for 15 minutes pretreatment

and culturing on ½ B5 medium with 2.5 cm length shoots can gave maximum

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(43 %) rooting response. Subcultured shoots rooted better during winter

period (December, January and February) similar to bud break response.

4. One tissue culture raised plant did not flower in the first year and produced

flower at two year of age after rooting, indicating the physiological maturity.

It is interesting and needs further research investigations.

B. Macropropagation in T. undulata:

1. Stem cuttings of mature T. undulata tree (15-16 year old) is greatly

influenced by season, position of branches in the tree and genotype. IBA do

not enhance rooting as compare to control.

2. Stem cuttings collected from upper part of the middle crown rooted

maximally (33.3 %) in the month of April raised in polybags under 80 ± 10 %

humidity 32 ± 5 oC temperature in one genotype.

3. Flowering also observed along with sprouting under mist polyhouse as they

carry the pre determined existing dormant flower buds.

C. Micropropagation in Ailanthus excelsa:

1. Bacterial contamination is serious problem in establishment of aseptic

cultures. Most of trees (90 %) near AFRI are having systemic bacterial

infection. Thus, it is sometimes difficult to establish contamination free

cultures from desired trees. In vitro cultures were established from one

genotype (male tree) of Ailanthus excelsa.

2. Addition of NAA or BA in the MS medium do not play significant role in bud

break similar to T. undulata. However, 10 mg/l Thiamine HCl added in the

medium promote shoot length.

3. Earlier work repeated on shoot multiplication, rooting and a few plants

produced. However, rooting is very difficult and unpredictable from mature

trees.

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The results of various experiments to standardize clonal propagation techniques

are discussed species-wise viz. T. undulata (5.1) and Ailanthus excelsa (5.2). In the

last part of discussion (5.3), present protocols are subjected to economic

consideration using methods described by Tomar et al. 2008 and further line of

research to minimize the cost of tissue culture plants.

5.1 Tecomella undulata:

Studies on micropropagation have been carried out by Rathore et al. (1991);

Bhansali (1993); Nandwani et al. (1995); Robinson et al. (2005) and Varshney and

Anis (2011). Whereas, no literature was available on macropropagation of

T. undulata till the time of initiating present macropropagation studies. Thus, the

discussion part of T. undulata is further divided into micro and macropropagation.

5.1.1 Micropropagation:

Micropropagation is preferred over macropropagation because of its

potential to multiply and produce large number of desired superior plants, through

out the year, which is a major obstacle in conventional approach (Watt et al. 1995;

Assareh and Sardabi 2005). Micropropagation process is divided into stage 1

(establishment of shoot culture), stage 2 (shoot multiplication) stage 3 (in vitro, ex

vitro rooting) and stage 4 (hardening & field trial). Micropropagation of mature trees

always have difficulties in establishing aseptic cultures, low shoot multiplication rate

and poor rooting as compared to seedling based micropropagation (Sutter and

Barker 1985; Sanchez and Vieitez 1991).

5.1.1.1 Stage 1 (Establishment of in vitro shoot cultures):

Establishment of aseptic shoot culture is a critical stage particularly when the

explants were excised from field grown plants (Hennerty et al. 1988; Savela and

Uosukainen 1994). Establishment of culture and further in vitro propagation depends

on the condition of plant material at the time of collection viz. green house material

or field grown plants and genotypes. Even the material (explants) collected from

single genotype represent extremes in topophysis (position), periphysis (local

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126

environment) and cyclophysis (age) effects. Mature tree explant possess the

problem of leaching of phenolic compounds in many of the tree species viz.

Azadirachta indica (Qurashi et al. 2004; Arora et al. 2010), Emblica officinalis (Goyal

and Bhadauria 2008) and Eucalyptus tereticornis (Joshi et al. 2003). In case of T.

undulata leaching was not at all a problem during the establishment of in vitro shoot

cultures.

In T. undulata maximum bud break was observed in January-February, which

differs from period reported by Rathore et al. (1991), where highest bud break was

achieved in the month of August-September. However, these two different results

have a gap of two decades though the location of experiments is Jodhpur

(Rajasthan). This difference may be due to some change in climate as observed in

last two decade in Jodhpur condition. Seasonal effect on bud break in mature tree’s

explants has been reported in M. esculenta (Bhatt and Dhar 2004), Acacia sinuta

(Vengadesan et al. 2002) and S. sebiferum (Siril and Dhar 1997). In all these species

late winter i.e. December to March is ideal for high percentage of bud break.

Luckily contamination was not a major problem in T. undulata and more than

75 % of stem nodal explants were established by using normal sterilization

procedures. Where as this is a serious problem in other studied species viz. Ailanthus

excelsa. Earlier workers also did not reported contamination as major problem in

T. undulata (Rathore et al. 1991; Robinson et al. 2005; Varshney and Anis 2011).

Highest contamination (23 %) was encountered with T. undulata during the rainy

season. This may be because of ideal temperature and high moisture in rainy season

which favours the growth of microbes. Similar results were obtained in Pyrus

pyrifolia (Thakur and Kanwar 2008), Arundinaria callosa (Devi and Sharma 2009),

Casuarina equisetifolia (Seth et al. 2007) and Banana (Josekutty et al. 2003).

However, the aseptic shoot cultures of T. undulata can be established throughout

the year unlike other species viz. Ailanthus altissima (Caruso 1974), Halesia and

Malus (Brand 1993), Melia azedarach (Husain and Anis 2009) and Ziziphus Spina-

christi (Assareh and Sardabi 2005).

Discussion

127

In the present investigation MS basal medium showed better responses for

bud break in nodal segments as compared to the B5 medium. Rathore et al. (1991)

also reported MS medium superior than other medium used in the same species.

The MS medium is having high concentration of nitrate, potassium and ammonia as

compare to other basal media. The second media used most frequently is B5 but the

levels of inorganic nutrients in the B5 medium are lower than in MS medium. MS

medium contain double the amount of nitrogen as compared to that of the B5 media

(Guru et al. 1999). Superiority of MS medium over SH, White's, Nitsch and B5

medium for shoot regeneration and establishment has also been observed in Agave

sisalana by Nikam (1997). MS medium is more effective for in vitro culture

establishment and subsequent multiplication in Papaver orientale (Zakaria et al.

2011), Melia azedarach (Husain and Anis 2009) and Acacia nilotica (Abbas et al.

2010).

In T. undulata, incorporation of plant growth regulators (BA, NAA & IAA) are

not showing significant effect on bud break but they are playing a positive role

towards shoots growth. Shoot length response was found better in NAA

supplemented medium than IAA. The effectiveness of BA in promoting in vitro

axillary shoot production in woody plant is well documented (Sahoo and Chand

1998; Nobre et al. 2000). Incorporation of NAA along with 2 mg/l BA was found

better than IAA because NAA is more, light and heat stable (Bonga and Van Aderkas

1992) and IAA readily oxidized by light (photooxidation) as well as by enzyme (IAA

oxidase). Similarly a combination of NAA (low concentration) and BA (higher

concentration) in MS medium was effective in Balanites aegyptiaca (Ndoye et al.

2003), Acacia auriculiformis (Girijashankar 2011), Aegle marmelos (Islam et al. 2007)

and Ailanthus excelsa (Sharma 1999).

The role of amino acids in growth and differentiation is known to a

considerable extent. Amino acids are important for growth regulation and as

modulators of growth and cell differentiation, which may be affecting general

metabolism and consequently morphogenesis (Basu et al. 1989). Amino acids are

taken up more rapidly by the plant cells, unlike inorganic nitrogen. Three different

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128

amino acids were incorporated in the medium for bud break and Arginine has shown

maximum bud break but no significant difference was observed on shoot length in

any of the treatment in T. undulata. Lowest bud break response was observed on

Lysine supplemented medium. Many of the researchers have tried different amino

acids for establishment, callus growth and somatic embryogenesis in many plant

species viz. Pelargonium (Wojtania and Garbyszewska 2001), Phoenix dactylifera (El-

Shiaty et al. 2004), Fragaria xananasa (Gerdakaneh et al. 2012).

5.1.1.2 Stage 2 (Shoot Multiplication):

Shoot multiplication through successive subculturing provide major benefit of

micropropagation over conventional methods. Due to this step tissue culture plants

can be produced through out the year and successive years. Establishing shoot

cultures initially multiply poorly with unpredictable rate but after few subcultures

gradually shoot multiplication rate is enhanced and stabilized.

Cytokinins play significant role in shoot multiplication and shoot growth.

Earlier workers, reported maximum shoot multiplication in MS + 0.01 mg/l IAA + 1.0

mg/l BA (Rathore et al. 1991), modified MS + 1 mg/l BA & modified Woody Plant

medium + 1 mg/l BA (Bhansali 1993), but their results were lacking proper statistical

analysis. Our experiments on shoot multiplication with cytokinin (BA) indicate that

shoot multiplication rate is higher at 2 and 4 mg/l BA but at these levels BA enhance

the callusing and reduces shoot length. Geetha et al. (1998) also reported that

explants of Cajanus cajan require 2.0 mg/l BA at the initial stage of shoot bud

regeneration and multiplication but further growth and proliferation of the shoot

was observed only after subculture to fresh medium with lower level of BA (1 mg/l).

Therefore, MS + 1 mg/l BA was recommended for shoot multiplication in

spite of low shoot multiplication than 2 and 4 mg/l in T. undulata. Similar results

were reported in many of the other species viz. Acacia senegal (Khafalla and Dafalla

2008), Paulwonia kawakamii (Lobna et al. 2008), Clitoria ternatea (Barik et al. 2007),

Azadirachta indica (Salvi et al. 2001), Lilium species (Takayama et al. 1991), Mentha

Discussion

129

spp. (Rech and Pires 1986), Withania somnifera (Sen and Sharma 1991) and

Pogostemon cablin (Kukreja et al. 1990).

Preparation of propagules by cutting them in different way and removing

unwanted callus, dead tissues and placing it on the fresh medium, play an important

role in shoot multiplication and their health (Memon et al. 2010; Udomdee et al.

2012). In T. undulata when shoot is cut into three parts viz. apical, middle and basal.

Shoot multiplication was poor in cultures derived from middle parts. Apical and basal

portion produced higher multiplication as compare to middle part. However, average

shoot length was highest with apical portions. Similar results were observed in many

species where an apical part of the propagule has shown higher length and low

shoot multiplication as compared to the basal portion viz. Ailanthus excelsa (Sharma

1999) and Paphiopedilum orchid (Udomdee et al. 2012).

Shoot multiplication subculturing period is finalized by recording and

analyzing the observations at fixed time intervals. Shoots were subculture when

growth of shoots stops increasing and before it is an adversely affecting their growth

due to non availability of sufficient nutrients. In T. undulata shoot multiplication rate

increased upto 20th day and shoot length upto 30th day. Therefore, 30 day (4 weeks)

subculturing period is sufficient before transferring these cultures on fresh medium.

Apical and basal parts of the shoots were found best over the middle part of

the propagule in the above experiment. Therefore, apical and basal part of the

propagule was placed on MS medium supplemented with and without 2 mg/l BA. In

present study basal and apical parts has shown highest increment in shoot number

and shoot length respectively on MS + 2 mg/l BA. But on comparing the results with

the previous experiment (table 14), it was observed that increasing the

concentration of BA was not found much helpful for higher shoot multiplication on

the contrary it has reduced the shoot length. Similarly higher concentration of BA

has negatively affected the shoot length in Pinus banksiana (Browne et al. 2001),

Anastasia (Al-Malki and Elmeer 2010) and Crepis novoana (Corral et al. 2011).

Therefore, it is suggested that the shoot can be multiplied on MS + 1 mg/l BA

medium. Whenever, the shoot multiplication rate goes down to a critical level during

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130

successive subculture, such shoots can be subculture on 2 mg/l BA medium to

enhance the multiplication rate. After achieving desired shoot multiplication again

MS + 1 mg/l BA medium can be used for subsequent subculturing to where rootable

shoots frequency is higher. Switching between these two BA concentrations during

successive subculturing is a management decision of production unit to maintain

consistent shoot multiplication and rootable shoots ratio.

Thiamine HCl (vitamin B1) is an essential co factor in carbohydrate

metabolism and is directly involved in the biosynthesis of some amino acids. Tissues

of most plants seem to require Thiamine HCl for growth (George et al. 2009).

Similarly, amino acids are primary nitrogen source and uptake can occur much more

rapidly than other form of inorganic nitrogen from the same medium (George et al.

2009). Therefore, Thiamine HCl and Glutamine were used in different concentrations

for improving shoot health and multiplication. In present studies, no significant

difference for shoot multiplication and shoot length due to these treatments were

observed. When the data of different concentration of Thiamine HCl was analysed,

regarding shoot length, it was observed that 26 % of the shoots were more than 2.5

cm length when cultured on 1 mg/l BA + 30 mg/l Thiamine HCl. This can be utilized

for production of shoots with increased length. Thiamine HCl has been used by many

of the researchers viz. Bonner (1937, 38), Robbins and Bartley 1937 (Tomato), White

1937 (Tomato), Polikarpochkina et al. 1979 (Zea mays), Barwale et al. 1986

(Soyabean), Chee 1995 (Taxus brevifolia) and Asano et al. 1996 (Zoysia japonica) for

healthy growth of shoots. Similarly Glutamine has been used by Bader and Khierallah

2009 (Phoenix dactylifera), Hamasaki et al. 2005 (pineapple), Vasanth et al. 2006

(Arachis hypogaea) and Shahsavari 2011 (Rice).

Shoots of T. undulata were multiplied on different concentration of sucrose

with the aim to find out the lowest concentration without much affecting shoot

multiplication to reduce the cost per plant to some extent. In the present study

lowering sucrose concentration below 3 % adversely affect the shoot length.

Therefore, 3 % sucrose in MS + 1 mg/l BA medium is the lowest concentration to get

sufficient number of rootable shoots as well as shoot multiplication in this species.

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131

Similarly 3 % sucrose was optimal in Spathiphyllum cannifolium (Dewir et al. 2006)

and Cajanus cajan (Geetha et al. 1998).

Ratio between number of shoots going to the rooting phase and shoots going

again to the subculturing phase finally decide the multiplication rate. While

calculating the multiplication rate both rootable and non rootable shoots were

included in the final multiplication rate which we have termed actual multiplication.

Our main aim is to get realistic shoot multiplication which is obtained by sum of

rootable shoots and the shoots kept for multiplication for next subculture divided by

the initial number of shoots used for multiplication. If after one cycle of subculturing

the number of non-rootable shoots (< 2.5 cm) is higher then these shoots will again

go for sub culturing. The excess of shoots will be waste when the number of shoots

subculturing reaches at the saturation capacity of the culture room. Thus the realistic

multiplication is desirable to a commercial tissue culture unit. A protocol is ideal

when realistic and actual multiplication is same. Thus further research must be

focused on improving realistic shoot multiplication.

5.1.1.3 Stage 3 (Rooting):

Success rate of in vitro or ex vitro rooting considerably influence the cost of

tissue culture plants. High frequency rooting was reported by Rathore et al. (1991)

by adopting two step procedure. This research paper was lacking experimental data

and statistical observation. Moreover, it was also not clear in this paper whether this

high rooting percentage was achieved with seedling material or with shoot derived

from adult trees. Thus, it was necessary to repeat these experiments and strengthen

these results.

Previous workers observed that auxin is required for rooting. Our results are

also in agreement with previous workers. In vitro multiplied shoots did not root on

MS medium lacking auxins (IBA). However, incorporation of IBA within media

resulted in inconsistent (lack of repeatability) and low percentage of rooting with

heavy callusing at the base of the shoots. This may be due to the mixed genotypes in

the stock culture and variation in relative humidity (RH) and temperature conditions

as shown in materials and methods (Graph 1).

Discussion

132

The ability of plant tissue to form adventitious roots depends on interaction

of many endogenous and exogenous factors. Auxins play important role in the root

formation in woody species, which are categorized difficult to root species in

literature viz. Apple rootstock, Castanea sativa, Pyrus calleryana, Juglans regia,

Simmondsia chinensis and Quercus petraea (Rugini et al. 1993). Different auxins like

NAA, IAA and IBA were tried in medium to induce rooting and only IBA was effective

in root induction of T. undulata. In many other species also IBA is preferred because

IBA is not easily destroyed by high temperature or light (Nissen and Sutter 1988).

Main advantage of using IBA over some other synthetic auxins is that IBA is

metabolized to IAA, i.e. the natural auxin (Epstein and Lavee 1984). IBA is being used

for the rooting of several tree species, (Shrivastava and Banerjee 2008; Martin 2002),

However, incorporation of IBA in the medium has increased the callusing and shoot

tip necrosis with very low percentage of rooting. In many other woody species also

rooting of in vitro raised shoots poses formidable problems viz. callusing, shoot tip

necrosis and leaf fall (Vieitez et al. 1989; Xing et al. 1997; Martin et al. 2007; Bairu et

al. 2009).

It is known that callusing during in vitro rooting can be reduced by lowering

salt concentration of medium, low concentration of auxin, two step method i.e.

treating the shoots with high auxin concentration for few minutes and transferring

them in hormone free medium and use of activated charcoal etc.

Among different (MS, B5, WPM and Hoagland) medium tried ½ B5 medium

was found best for rooting (%). Similarly B5 was found better for rooting in P.

orientala (Zakaria et al. 2011) and Argyrolobium roseum (Khanna et al. 2006). It is

well known that low salt concentrations favours rooting and B5 medium has low salt

levels than in MS medium (Guru et al. 1999).

Treatment of shoots in vitro with high concentration (100-200 mg/l) of IBA

for 15 minutes and subsequent transfer to hormone free medium was beneficial for

quality rooting as well as over coming the callusing problem in T. undulata. These

results are similar with the observation of Rathore et al. 1991. But our results differ

in the percentage of rooting. Rooting was never reached to 60-70 % in our

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133

experiments. Moreover, rooting was unpredictable while repeating the experiments.

It appears that annual pattern of culture room conditions (temperature and RH) has

influence of ambient conditions inspite of control systems, which are also dependent

on electricity. Therefore, failures of electricity are major cause of weak control

system of culture room conditions. This problem needs to be addressed in future

research projects on this species.

A combination of IBA and NAA was much effective than individual auxin. High

auxin dip treatment for small duration (15 minutes) was adopted to reduce the

callusing, shoot tip necrosis and to improve the rooting percentage. Similar work has

been done in Achras sapota (Purohit and Singhvi 1998), Annona squamosa (Nagori

and Purohit 2003), Rice cultivars (Wahyuni et al. 2003), Wrightia tinctoria (Purohit

and Kukda 2004), Apple rootstock (Sharma et al. 2007) and Vicia narbonensis (Kendir

et al. 2008).

Problems faced during in vitro rooting by direct method were shoot tip

necrosis (100 %) and leaf fall (100 %) in different experiments. Addition of Silver

nitrate and Activated charcoal decreased the callusing and shoot tip necrosis in

Tecomella undulata to certain extent. Accumulation of ethylene has been reported

as the cause of necrosis and abscission of leaves and shoots (Martin 2002). Shoot tip

necrosis causes severe loss of cultures in several woody species (Kataeva et al. 1991;

Hammatt and Ridout 1992; Sita and Swamy 1993; Amo-Marco and Lledo 1996; Xing

et al. 1997; Kulkarni and D’Souza 2000). Silver nitrate (AgNO3) has been known to

inhibit ethylene action (Beyer 1976 a). Silver ion is capable of specifically blocking the

action of exogenously applied ethylene in classical responses such as abscission,

senescence and growth retardation (Beyer 1976 c). Addition of AgNO3 to the culture

media greatly improved the regeneration of both dicot and monocot plant tissue

cultures (Beyer 1976 b; Duncan et al. 1985; Davies 1987; Purnhauser et al. 1987;

Songstad et al. 1988; Chi and Pua 1989; Veen and Over Beek 1989; Bais et al. 2000;

Giridhar et al. 2003). The exact mechanism of AgNO3 action on plants is still unclear.

However, few existing evidences suggest its interference in ethylene synthesis

mechanism (Beyer 1976 b). AgNO3 has been employed in tissue culture studies for

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134

inhibiting ethylene action because of its water solubility and lack of phytotoxicity at

effective concentrations (Beyer 1976 a).

In the present study activated charcoal has reduced callusing and shoot tip

necrosis to some extent but it has negatively affected the in vitro rooting in

Tecomella undulata. Activated charcoal used in nutrient media has an adsorption

preference for moderately polar rather than apolar or highly polar organic

compounds. They show greater adsorption for aromatic than olefinic unsaturation

products (Yam et al. 1990). Therefore, aromatic compounds such as the phenolics

and their oxidates, auxins (IAA, NAA & IBA), cytokinins (BA), and hormones could

have great adsorption affinity for activated charcoals. Activated charcoal provides a

degree of darkness during in vitro development of shoots. Light is a major factor of

the culture environment and has been shown to have an effect on organized

development under in vitro conditions (Pan and Staden 1998). Similarly Van (1987)

and Phoplonker and Galigaripd (1993) reported the inhibition of callus formation due

to activated charcoal in Beta vulgaris and Lupinus mutabilis respectively. Webb et al.

(1988) observed that activated charcoal inhibited rhizogenesis when included in the

rooting medium. Activated charcoal had a marked negative effect on the percentage

of rooted shoots of Prunus silicina also (Rosati et al. 1980).

Thiamine HCl (vitamin B1) is important constituent of many culture media

and necessary for root growth. Bonner (1952) reported that Thiamine is synthesized

in the leaves and transported to the roots. The vitamin B complex is known to

stimulate cell division (Jablonski and Skoog 1954) and Ascorbic acid used as an

antioxidant agent can reduce browning of medium resulted due to exudation of

phenolic compounds from mature explants and their oxidation. Therefore, it

prevents necrosis also being an antioxidant in nature (Rumary and Thorpe 1984,

Gupta 1986). In the present study, medium incorporated with Thiamine HCl has

improved rooting percentage and Ascorbic acid has favoured maximum number of

roots/shoots. Our results are in agreement with the previous literature, which

suggest that Thiamine is important for root growth (Chee 1995). Excellent root

development due to Thiamine HCl was observed in Matteucia struthiopteris

Discussion

135

(Dykeman and Cumming 1985), Ostrich fern (Bonner and Devirian 1939), T. brevifolia

and T. cuspidata (Chee 1995). It has also been reported by Bose et al. (1982), that

application of Ascorbic acid in combination with an auxin (IBA) promotes rooting in

terms of number of roots/cutting in various plant species. Ascorbic acid makes

rooting earlier and improves the quality of roots as compared to those treated with

auxin alone (Sharma and Rai 1993; Cong-Linh Le 2001).

Annual pattern of relative humidity (RH) and temperature inside the culture

room revealed variations in both parameters during different months of the year

(Graph 1). When the results of all rooting experiments conducted in different

months analysed it was noticed that optimal rooting was recorded in winter period

only i.e. December, January, February and March when the average temperature

was 24 ± 1 oC and RH 32 %. It indicates that the culture conditions influence the

rooting procedure. In this species similar annual pattern of bud break in nodal

explants is also observed. These bud break and rooting annual pattern need to be

studied further. These patterns under in vitro conditions may be due to carrying

memory of physiological adoption of this species with ambient annual condition of

Rajasthan. Another reason may be due to the effect of varying temperature and RH

annual pattern of culture room.

Data analysis with different shoot length used for rooting experiment

revealed that shoots less than 2.5 cm length did not root. Similarly in Prosopis

cineraria, shoots greater than 2.5 cm length rooted in MS medium supplemented

with 3.0 and 5.0 mg/l IBA (Kumar and Singh 2009). Rhizogenesis was effectively

induced in well-elongated shoots (> 4 - 6 cm) in Argyrolobium roseum (Khanna et al.

2006). A group of scientists suggested that elongation has been a preparatory stage

for rooting during which the carry over effect of cytokinins is reduced (Druart et al.

1982; Gulati and Jaiwal 1994; Khanna et al. 2006), which resulted in better rooting.

5.1.1.4 Stage 4 (Hardening):

Plantlets of Tecomella undulata were produced via tissue culture using nodal

explants from mature tree. The plants were successfully hardened and planted in

earthen pots. One of the micropropagated plant developed flowers at the age of two

Discussion

136

years only. The early flowering, of tissue culture plants is not desirable for clonal

propagation but interesting to plant breeders, physiologist and molecular biologist.

Early flowering in tissue culture plants have also been observed in Betula pendula

(Huhtinen and Yahyaoglu 1974), Pinus sylvestris (Haggman et al. 1996) and

Dendrocalamus asper (Sharma 2011).

Hardening success in any species is highly dependent on quality and healthy

rooting. Since in present investigations rooting step could not be improved in terms

of quality and quantity rooting, proper experiments were not feasible on hardening.

Efforts were made to harden the plants ever produced as an output of rooting

experiments. Rooting was observed in many of the treatments. But poor health of

the rooted shoots and shoot tip necrosis, was the major obstacle to provide

sufficient plants needed for hardening experiments. Approximately 40 % of the

plants produced through in vitro rooting were found healthy, which were used for

hardening. Scientist have focused their research on hardening/acclimatization of

tissue culture plants to understand and improve the protocols efficiency (Fabbri et

al. 1986; Drew 1992; Deng and Donnelly 1993; Bolar et al. 1998; Pospisilova et al.

1999; Hazarika 2000; Deb and Imchen 2010; Kaur et al. 2011).

Due to unavailability of more than 15 plants at a time, no hardening

experiments were conducted only basic hardening procedure was followed. Initially

the plantlets were hardened in vitro inside the culture room for one month because

the plants can not be directly transferred to the mist polyhouse as the internal

environment of the polyhouse was mainly kept for macropropagation which was not

ideal for tissue culture plants. Plantlets inside culture room were observed daily and

after attaining certain growth these plantlets were transferred to the mist polyhouse

as described in materials and methods. Similar, procedure was adopted by many

researchers in Sterculia urens (Purohit et al. 1994), Wrightia tomentosa (Purohit and

Dave 1996) and Annona squamosa (Nagori and Purohit 2004).

There has been plenty of research work for optimization of different stages

upto rooting of the plant species but there is lack of research work associated with

the hardening of these plants particularly in case of trees. Overall success of the

Discussion

137

tissue culture protocol at commercial level depends on the number of healthy plants

produced after hardening and fit field plantations. Therefore, there is need to

emphasize the research on rooting and hardening of this species in particular and in

several other species.

5.1.2 Macropropagation:

Macropropagation is simple, economic and easily adoptable by forest

department as compare to highly technical micropropagation. Macropropagation

includes rooting of stem cuttings, grafting, budding and air layering.

Macropropagation through stem cutting is a common method used in propagating

many tree species viz. Acacia nilotica (Goel and Behl 2005; Negi 2001), Acacia

senegal (Dantu et al. 1992), Azadirachta indica (Ehiagbonare 2007), Dalbergia sissoo

(Singh et al. 2011, Husen 2008), Eucalyptus camaldulensis (Karthikeyan and Sakthivel

2011) and Tectona grandis (Palanisamy et al. 2009). However, some trees like

Popular, D. sissoo etc. can be propagated easily but many trees are still difficult to

propagate vegetatively viz. Acacia nilotica (Goel and Behl 2005; Negi 2001), Prosopis

juliflora (Goel and Behl 2005), Ailanthus excelsa (Sharma 1999) and Aesculus indica

(Majeed et al. 2009). Tecomella undulata comes under the category of recalcitrant

root species. In the present thesis efforts were made to study the factors affecting

rooting process in Tecomella undulata mature trees.

Auxins play an important role in inducing rooting or enhancing rooting

response in many tree species viz. E. saligna, E. globulus (Fett-Neto et al. 2001),

Triplochiton scleroxylon (Leakey et al. 1982), Tectona grandis (Husen and Pal 2006,

2007) and Aesculus indica (Majeed et al. 2009). Requirement of auxin treatment for

rooting also depend on juvenility and physiological status of stem cuttings (Rema et

al. 2008; Osterc et al. 2009). Surprisingly, auxin (IBA) does not play any beneficial

effect on rooting of T. undulata stem cuttings. In fact IBA treatment was inhibitory

for rooting in this species. Trueman and Peters (2006) also reported that application

of IBA at different concentrations did not accelerate root protrusion or affect final

rooting percentages in Wollemia nobilis. Similarly, the stem cuttings of P. azorica

(Moreira et al. 2009), ‘Gisela 5’ dwarfing cherry rootstock (Stefancic et al. 2005) and

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P. salicina (Neto 2006) did not beneficially affected by IBA treatment. Exogenous IBA

is generally applied at the base of the cutting and basal application of auxin in

difficult to root species may not lead to an increase in auxin concentration due to

lack of auxin receptors in these cells that would give rise to adventitious root

formation (Ford et al. 2002). Furthermore, it is known that exogenously applied

auxins, in those seasons when endogenous levels are high due to high meristematic

activity, can bring the total auxin concentration to supra optimal levels in such trees,

e.g. Populus (Nanda and Anand 1970) and Olea (Ansar et al. 2009; Moreira et al.

2009).

Polybags were found better for rooting in T. undulata stem cuttings as

compared to root trainers. In Albizia procera (Gera et al. 2000) and Leucaena

leucocephala (Ferdousee et al. 2011) polybags were found best in terms of growth

and germination. Similarly in Hevea brasiliensis larger size of the polybags has shown

positive effect on root growth and helped in better survival (Varghese et al. 2005).

Larger size of the poly bags allow the cuttings to survive, sprout and root even after

2-3 months, with more availability of nutrients as compared to root trainers in T.

undulata. But in contrast to our results, many of the researchers reported root

trainer best, in Hevea species (Soman et al. 2011), Bamboo species (Gera et al. 2007)

and Indian Sandalwood (Annapurna et al. 2004) because improper root system was

observed in polybags.

No rooting was observed in any of the potting mixtures tried, but highest root

primordia formation was observed in sand. The potting mixture should have good

aeration, water and nutrient holding capacity. Sand used as potting mixture has good

aeration but poor water and nutrient holding capacity (Nebel and Wright 1993).

Gautam et al. (2010) found vermiculite good for short term use but after 45-50 days

it showed poor aeration and nutrient deficiency in the media. This may be the

reason of no rooting response in any of the potting medium. There is need of further

investigation to standardize the potting mixture in T. undulata. Many of the

researchers have combined various potting mixtures to get optimal results In

Santalum album sand: soil: compost (40 : 10 : 50) found best for root shoot ratio

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(Annapurna et al. 2004). Similar work for standardization of potting mixture has

been done by various workers in Acacia catechu (Nandeshwar and Patra 2004),

Acacia auriculiformis (Sharma et al. 2004) and Perlagonium hortorum (Mamba and

Wahome 2010) and Acacia albida (Harsh and Muthana 1985).

High rooting percentage was obtained in the stem cuttings kept inside

polyhouse. This experiment was conducted with the aim to know the rooting

potential of T. undulata stem cuttings in outside polyhouse conditions. Similarly in

Mulberry genotype, stem cuttings kept inside polyhouse resulted in enhanced

rooting ability (Baqual et al. 2012). The high temperature and humidity inside

polyhouse is favourable to induce rooting in stem cuttings. Similarly in Hevea

brasiliensis (Mercykutty et al. 2012) and Quercus glauca (Purohit et al. 2005) plants

produced inside polyhouse through budding and air layering respectively have

showed better health as compared to plants grown outside polyhouse.

It was needed to understand the growth and physiological behaviour of trees,

to know the effect of tree physiology on some of the common practices applied in

mature tree management. Aging in trees are of three types chronological,

ontogenetical and physiological but rejuvenation is opposite of aging. Rejuvenation

is also of two types ontogenetic and physiological. Through physiological

rejuvenation methods the aging process can be slow down. Pruning methods viz.

pollarding, lopping and coppicing are part of physiological rejuvenation (Del Tredici

1998). Stem cuttings were collected from differently managed trees i.e. lopped,

pollard and coppice shoots and kept for rooting experiments. Highest sprouting and

root primordia formation was observed in pollard shoots and no rooting was

observed in any of the cuttings. Lopping in Prosopis cineraria tree has improved

height, growth and stem diameter (Sharma and Gupta 1981). Plenty of literature is

available on influence of physiological status of cuttings on rooting potential (Puri

and Khara 1992; Dassanayake et al. 2000; Yeboah et al. 2009). Further research work

is needed in future on T. undulata, regarding this aspect.

In many species thickness of stem cuttings influences rooting response viz.

quality and quantity of roots due to differences in carbohydrate reserves (Hartmann

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et al. 2002). Stem cuttings of T. undulata, ranging from 1.0 to 1.5 cm thickness were

rooted. Stem cutting with lower or higher thickness than this middle range (1.0-1.5

cm) did not root. The poor performance of thin cuttings is also attributed to the

reason that the cuttings are still under maturity and may be devoid of sufficient food

material for induction of roots and shoots. Reserve food material plays a vital role in

root shoot induction and growth (Nanda et al. 1972; Haissig 1974). The under

performance of the larger sized cuttings may be attributed to the reason that these

cuttings are more woody and might have converted most of the food material for

the lignifications which resulted in over lignified stem and caused poor or no rooting.

Stem cuttings of middle range thickness (2.5-3.0 cm) were found suitable for rooting

as compared to other types of cuttings in Jatropha curcus (Kathiravan et al. 2009),

Prunus cerasus × Prunus canescens (Exadaktylou et al. 2009) and Ficus species

(Mathew et al. 2011).

Season has marked effect on rooting of stem cuttings (Singh et al. 2004). In

present studies time span from January–March (late winter) period is found best for

rooting in stem cuttings of T. undulata. Before onset of leaf fall trees generally

accumulate nutrients in the shoots which are subsequently utilized for emergence of

new sprouts (Palanisamy et al. 1998). The formation of new sprouts leads to

elevation of endogenous root forming substances including auxins (Went 1929;

Bouillenne and Went 1933; Avery et al. 1937). The shoot collection during the

dormant season is important for rooting of dormant cuttings because the shoots

must pass through an adequate chilling period of physiological dormancy (rest)

before rooting will commence (quiescence) (Coleman et al. 1993; Chandler and

Thielges 1973; Nanda and Anand 1970). The variation in seasonal rooting response

may be attributed to the physiological condition of the plant cuttings. Cellular

activities during root initiation require availability of sugars which are synthesized

due to activity of various hydrolytic enzymes (Nanda 1975). The activity of these

enzymes might have been at the highest level during monsoon and post monsoon

months. The failure of cuttings to produce good root system in non-monsoon

months may be due to a high rate of metabolism and increased inhibitor–promoter

ratio (Eganathan et al. 2000). Many stem cuttings collected during the winter season

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has showed highest rooting and survival rate viz. P. oceanica (Balestri and Lardicci

2006), P. deltoides x P. nigra ‘DN17’ (Smith and Wareing 1974), Populus balsamifera

L. (Cunningham and Farmer 1984), P. deltoides (Zalesny and Wiese 2006), Pongamia

pinnata (Rangan et al. 2010) and Bitter Almond hardwood cuttings (Kasim et al.

2009).

It has been reported in many woody species that genetic make of individual

genotype also influence rooting response through stem cuttings. In the present

investigation among four different genotypes tree no. 9 has shown maximum

rooting. Differential rooting of different genotypes have been observed in many

other plant species viz. Laucaena leucocephala (Shi and Brewbaker 2006), Cercis

canadensis (Wooldridge et al. 2009), Eucalyptus camandulansis, E. teriticornis

(Verma et al. 1993), A. auriculiformis (Haines et al. 1992), Salix planifolia (Houle and

Babeux 1983) and Pongamia pinnata (Rangan et al. 2010).

Rooting response of stem cuttings can be affected by position on the mother

tree from where it was collected in spite of being genetically identical (Olesen 1978).

The difference in the characters of part of the tree, which are determined by

position is termed “Topophysis”. The effect of topophysis (position) on rooting of

cuttings was reported in many species viz. Pinus radiata (Libby and Hood 1976),

Tectona grandis (Nautiyal et al. 1992) and Delbergia sissoo (Ansari et al. 1995). This

effect on rooting was also observed in T. undulata. Stem cuttings collected from

upper part of the branches belonging to middle crown rooted better. In case of tree

species, the degree of juvenility is inversely proportional to the distance (along the

trunk and branches) between the root shoot junctions and branches (Razdan 1993).

The endogenous auxin levels decreases as the distance from the apices of the

branches within the same plant increases (Overbeek 1938). In most tree species

rooting ability of cuttings has been reported to increase from apical to basal part of

the crown/shoots which has been attributed to accumulation of carbohydrates at

the base of shoot (Hartmann et al. 1997). Cuttings taken from the middle position

had the best rooting percentage followed by apical and basal positions in many

species viz. Tectona grandis (Husen and Pal 2007), Prunus cerasus × Prunus

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canescens (Exadaktylou et al. 2009) and Dalbergia sissoo (Husen 2004). Therefore, it

is evident from these findings that optimal branch positions for the best rooting

percentage vary with the plant species. The effect of position on rooting may be

caused by variation in the physiological status of shoot/cutting tissues on stock

plants resulting in occurrence of gradients along the stem axis in the cellular activity

or in the level of assimilates or growth regulators or in the level of lignification etc.

(Hartmann et al. 1997).

5.2 Ailanthus excelsa:

Ailanthus excelsa is another tree species which is difficult to propagate by

vegetative means. Very poor success has been achieved through stem cuttings

(Sharma and Tomar 2003). Considerable success has been achieved in

micropropagation through seedling explant (Sharma 1999). However, success was

limited through explants excised from mature trees. Rooting of excised shoots

derived from seedlings as well as coppice shoots (Sharma 1999). Present studies

were aimed to improve the micropropagation technique in Ailanthus excelsa.

Various experiments were conducted on shoot establishment, multiplication and in

vitro, ex vitro rooting. In the previous literature Sharma (1999), reported that

individual male and female trees of Ailanthus excelsa are present in nature.

Therefore, we have selected two male and two female trees for the present study.

5.2.1 Stage 1 (Establishment of in vitro shoot cultures):

Difficulty in establishing shoot cultures from mature trees has been reported

by (Sharma 1999). Shoot cultures were established only from explants excised from

coppice shoots with great difficulty. The major problem encounter in establishing in

vitro cultures was bacterial contamination. Due to unavailability of coppice shoots

through out the year, annual pattern of bud break was not studied but it was

observed that bud break percentage was found better in the month of April and

May. Four trees (2 male and 2 female) were selected for collecting coppice shoots. In

vitro shoot cultures could be established from only one male plant growing inside

vegetative propagation complex in AFRI campus. In rest of the three plants heavy

bacterial contamination was recorded within one week. Therefore, efforts were

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143

made to establish contamination free cultures. It was experienced that plant

hormone did not enhanced bud break in Ailanthus excelsa.

Establishment of aseptic culture in Picea glauca (Ellis et al. 1989) and

Eucalyptus urophylla × Eucalyptus grandis (Ouyang et al. 2012) has been achieved by

supplementing antibiotics in the medium. Two antibiotics namely Levofloxacin and

Ciprofloxacin were used in different concentrations to remove the bacterial

contamination. Bacterial contamination was reduced to some extent but no bud

break was achieved in the medium containing both the antibiotics.

Another experiment was conducted with Plant Preservative Mixture to

reduce the contamination. Plant Preservative Mixture (PPM) is a mixture of two

Isothiazolones viz. Methylchloroisothiazolinone and Methylisothiazolinone. It is

effective against both bacteria and fungi. It is also heat stable unlike other

conventional antibiotics, this can be autoclaved in the media. These characteristics

of plant preservative mixture make it an attractive alternative to conventional

antibiotics and fungicides in plant tissue culture (George and Tripepi 2001). In the

present study establishment of cultures from the nodal segments was very difficult

due to bacterial infection. Use of plant preservative mixture has reduced the

problem to some extent. Bud break was also achieved through the nodal segments

without bacterial infection. The difficulty in establishment of contamination-free in

vitro cultures was also reported by many groups (Salvi et al. 2002; Shirgurkar et al.

2001; Sunitibala et al. 2001; Naz et al. 2009). The supplementation of plant

preservative mixture in medium was also proved helpful in getting contamination

free cultures of Curcuma longa (Naz et al. 2009) and Petunia hybrida (Miyazaki et al.

2010).

5.2.2 Stage 2 (Shoot multiplication):

Establishment of cultures was very difficult due to the bacterial

contamination and only few cultures from the male tree of A. excelsa were survived.

The aseptic cultures were maintained by repeated subculturing on MS + 1 mg/l BA

medium as described by Sharma (1999) and experiments were carried out using

Glutamine and Thiamine HCl in medium to improve multiplication and shoot length.

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In A. excelsa Thiamine HCl has shown significant difference on shoot length

but application of different concentrations of Glutamine have not shown any

significant effect on growth. Similar results were also observed during shoot

multiplication of Tecomella undulata (present thesis page 82, 83). Rao and Prasad

(1991) mentioned that the amino acid L-Glutamine increases shoot bud regeneration

and Vengadesan et al. (2002) who found that it is ideal for shoot bud induction in

Acacia catechu and A. nilotica. Also, Mathur et al. (2002) reported that incorporation

of additives as Glutamine and Thiamine HCl was found to be most effective in shoot

elongation as well as accelerating multiple shoot proliferation. Although green plant

parts normally synthesize thiamine, additional amounts to the culture medium

appeared to stimulate explants growth and may enhance root growth in the rooting

stage (Hegazi and Gabr 2010).

5.2.3 Stage 3 (Rooting):

Long shoots (1.5-4.0 cm) of Ailanthus excelsa were selected for in vitro and ex

vitro rooting experiments conducted in culture room and polyhouse respectively.

Short duration (15 min) treatment of auxins solutions to shoots subsequently

cultured on auxin free medium did not worked in A. excelsa. Whereas the same

method (pretreatment of shoots with auxin) was successful in rooting of T. undulata

shoots. Direct incorporation of auxins like IAA and IBA also failed to induce rooting in

Ailanthus excelsa. Only NAA (1.5 and 2 mg/l) in the medium could induce poor

rooting (up to 15 %). Sharma (1999) reported 50 % on MS + NAA medium. Present

rooting results are done with shoots derived from male plants. Where as this

information is lacking in previous research work (Sharma 1999). This could be the

reason in the significant difference in rooting on same medium. Superiority of NAA

over IAA and IBA for rooting in mature trees is reported in Strobilanthes

hamiltoniana (Shameer 2006) and Prunus persica L. (Nagaty 2012).

5.2.4 Stage 4 (Hardening):

Hardening is essential for the better survival and successful establishment of

the in vitro raised plantlets. Due to unavailability of more than 15 plants at a time, no

hardening experiments were conducted, only basic hardening procedure was

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145

followed as described earlier in materials and methods and used in T. undulata.

Hardening is found very difficult in this species as reported previously by Sharma

(1999).

In this species major challenges are establishment of contamination free

cultures, high frequency rooting and hardening. Comparative studies of male and

female plants under in vitro conditions also needed during different stages of

micropropagation. Serious efforts are required with different approaches to

overcome these problems.

5.3 Economic Consideration:

Micropropagation is a high-tech method of vegetative propagation by which

selected genotype can be rapidly mass multiplied aseptically under controlled

environmental conditions (Murashige 1974). This process consists of four stages, (1)

Establishment of aseptic cultures, (2) Shoot multiplication, (3) Rooting of shoots and

(4) Hardening. The primary advantage of micropropagation is the rapid production of

quality, disease-free and uniform planting material. The plants can be multiplied

continuously throughout the year, irrespective of the season and weather (Murashige

1974). However, micropropagation technology is an expensive one as compared to

conventional methods and requires several types of skills. It is a capital-intensive

industry.

As with any other technology, the success of tissue culture within the nursery

industry will be determined by economic factors (Lineberger 2009).

Micropropagation protocols are acceptable in business when they are really

profitable. Many plant tissue culture protocols are commercialized particularly of

ornamental plants. However, plant tissue culture is not equally successful in case of

woody species. Micropropagation protocols of adult and mature trees were classified

into different levels on the basis of their efficiency by Maynard (1988), which

influence the cost of tissue culture plants. Therefore, economic criteria must be

applied on micropropagation protocols of woody species before recommending them

for commercialization. Cost analysis of tissue culture plant protocols is needed and

accordingly future research is directed to reduce cost at level of market demand.

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146

Micropropagation protocol efficiency is an indicator of low cost plant

production and it is actual rate of producing plants per unit time (year) through a

defined set up (tissue culture unit). This tissue culture unit comprises infrastructure

and manpower. Tomar et al. (2008) developed a method of tissue culture plant's cost

calculation with a defined set up. Same method is adopted for cost calculation of

Ailanthus excelsa and Tecomella undulata plants using level of protocols developed in

present thesis.

But before going for actual cost analysis of present protocols for above

mentioned two species, it is necessary to understand each step contribution on cost.

It is also necessary to point out here that failure of poor success in latter stages

contributes more than former stages because the plant lost at a stage add its cost to

the remaining plants. So the age of dying propagule/plant is higher it will augment

accordingly higher cost to remaining plants. Stage first establishment of shoot

culture contribute minimum to plant produced at the end of all steps as compare to

later steps. Second step is unique and important in micropropagation, which makes

it advantageous over conventional methods. This step is called shoot multiplication

and in vitro shoots can be multiplied at regular interval of time. This step contributes

a lot on rate of plant production at regular interval of time and hence needs to

understand carefully.

Shoot multiplication is calculated by dividing total number of shoots at end

subculture period with total number of shoots at the commencement of subculture.

This we can call actual shoot multiplication (ASM). ASM is used by majority of the

scientist involve in developing plant tissue culture protocols. However, when it

comes to economics of tissue culture plants it will not provide correct economics.

ASM is inferior method for a manager required for estimating or preparing a plant

production schedule with a protocol up to certain period of time. It is bound to

happen at each subculture, some shoots has to be discarded due to limiting space in

culture room. Obviously, in this situation only injured, abnormal and infected shoots

will be discarded and only healthy shoots will be preferred for subculturing. Hence,

the realistic shoot multiplication (RSM) is calculated here by dividing sum of rootable

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147

shoots and selected healthy shoots at end subculture by total initial number of

shoots at the time of subculturing.

Therefore, RSM always will be smaller and hardly ever equal to ASM. The

best situation is when RSM is equivalent to ASM. Realistic shoot multiplication of

Tecomella undulata in table 14a is 1.78, whereas actual shoot multiplication is 2.43.

Cost Calculations:

The cost per plant is estimated with the help of equation and method

described by Tomar et al. (2008) for tissue culture plants. This method takes into

account all the stages of protocol from in vitro establishment to the plantable sized

plants after hardening and the cost evolve on chemicals, glassware, electricity,

manpower, equipments etc. on annual basis. The cost of T. undulata is calculated as

per the present protocol with the actual shoot multiplication rate of 2.9 (which is

calculated from repeated long-term subculturing data), 40 % rooting and 35 %

hardening success. Estimated production of tissue culture plants using above

protocol with defined facilities and manpower will be 27,104 plants in first year with

a cost Rs. 65.17 per plant. However, in second year the cumulative production (first +

second year) will be 69,328 plants and cost will also reduced to Rs. 51.0 per plant.

The cost will be higher through realistic shoot multiplication. The realistic shoot

multiplication rate in the present protocol is 1.78 derived from one of the

experiments where the shoot multiplication is lowest rate. If we consider the

realistic shoot multiplication rate (1.78) for the above protocol, estimated

production will be 17,068 plants at rate of Rs. 103.5 per plant in the first year and

42985 plants at rate of Rs. 82.18 in the subsequent year. The desirable protocol at

commercial level should have higher RSM (above 4.0 fold), rooting (above 80 %) and

hardening (above 80 %) values. On assuming the realistic shoot multiplication rate of

4.0, 80 % rooting and 80 % hardening success the cost per plant in the first year will

be 10.45 with 1,68960 number of plants and rupees 8.12 in the second year with

4,35200 number of plants.

In case of Ailanthus excelsa we have calculated the cost per plant from the

previous work done by Sharma 1999 with seedling material. The protocol include

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actual shoot multiplication (ASM) rate of 4.3, 50 % rooting and 10 % hardening

success. Accordingly the cost in first year is 124.7 with 14160 plants and 96.7 with

36520 numbers of plants in the second year. Preset protocol of Ailanthus excelsa for

male tree has 1.3 ASM, 15 % rooting and 25 % hardening success. Accordingly, first

year tissue culture plants production will be 3420 at the rate of Rs. 516.5 per plant

and in subsequent years will be 8490 plants at the rate of Rs. 416.1 per plant.

Both protocols need to be improved at the targeted scale hence efforts are

needed to study further and more emphasis may be given on rooting and hardening.

Realistic shoot multiplication should be calculated for a commercial protocol. In case

of Ailanthus excelsa tissue culture protocols are required for female plant only as it

has better growth characteristics for fodder and wood (Tomar 2012).