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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
Discussion
124
(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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125
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
Discussion
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
Discussion
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
Discussion
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.
Discussion
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
Discussion
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
Discussion
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
Discussion
139
(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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140
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
Discussion
141
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
Discussion
142
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
Discussion
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.
Discussion
144
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
Discussion
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.
Discussion
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
Discussion
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
Discussion
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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).
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