Improving regeneration in Shisham ( Dalbergia sisoo Roxb.

By

Arzoo Rubab

2017-ag-3625

B.S Molecular Biology and Biotechnology

In partial fulfillment of the requirement for the degree of

MASTER OF PHILOSOPHY IN BIOTECHNOLOGY

Centre of Agricultural Biochemistry and Biotechnology (CABB)

FACULTY OF AGRICULTURE

UNIVERSITY OF AGRICULTURE, FAISALABAD

2019
 DECLARATION

I hereby declare that the contents of this thesis, “Improving regeneration in Shisham
(Dalbergia sisoo Roxb.)” are product of my own research and no part has been copied
from any other published source except the references, standard mathematical, genetic
models, equations, formulae, protocols etc. I further declare that this work has not been
submitted for the award of any other degree/diploma. The university may take action
if the information is somehow incorrect.

Arzoo Rubab

Reg. No. 2017-ag-3625

The Controller of Examinations,

University of Agriculture,

Faisalabad.

We, the supervisory committee certify that the contents and form of thesis
submitted by Arzoo Rubab, Regd. No. 2017-ag-3625 have found satisfactory and
recommended that it be processed for evaluation by external examiner for the award of the
degree.

Supervisiory committee

Chairman ______________________
Dr. Iqrar Ahmad Rana

Member ___________________________
Dr. Adnan Khan Niazi

Member ___________________________
Dr. Muhammad Kashif
 DEDICATION

I dedicate this effort to

My Graceful, Loveable Father

Who is true inspiration for me and

Always seem to surround my life.

 ACKNOWLEDGEMENT

All praises and thanks to ALMIGHTY ALLAH, Who bestowed mankind with
knowledge and wisdom. All the respect and honors to Holy Prophet Muhammad, a star
brightening the path of faith and knowledge. Who enabled us to recognize our creator
and declared it to be obligatory duty of every Muslim to acquire knowledge.

No acknowledgement could ever adequately express my obligation to my Father,

Altaf Hussain and my esteemed affectionate Mother, for my success is fruit of their
support and prayers.

It is a great privilege for me to record my cordial thanks to kind supervisor Dr.

Iqrar Ahmad Rana, Assistant Professor, Centre of Agricultural Biochemistry and
Biotechnology (CABB), University of Agriculture, Faisalabad for his sincere co-operation,
guidance, and inspiring attitude made it very easy to undertake this project toward its
successful accomplishment.

I offer my earnest gratitude to members of supervisory committee Dr. Adnan

Khan Niazi and Dr. Muhammad Kashif.

A great contribution through good wishes, moral support, and general help comes
through my friends Muhammad Haseeb Ali, Shahid Ali, Babar Shehzad,
Muhammad Mehran Abbas, Danish, Sayyad Hammad Mahboob, Syed Naeem
Sajid, Imtiaz Hussain, Iqra Mohiuddin, Amna Asghar, Nadia Azhar, Shehr
Bano. Similarly, most affectionate seniors Somia Ilmas and Aqsa has always
provided me all possible guidance and I am thankful to them for their limitless
kindness. May Allah bestow strength to all these splendid personalities (Ameen).

Arzoo Rubab
 Contents
Chapter Title Page
No. No.

Abstract

1. Introduction 1

2. Review Of literature 8

3. Materials and Methods 39

4. Results and Discussion 54

5. Summary 76

References 77

1
 List Of Tables

Sr. No. Title Page No.

3.1 Recipe of the Half MS culture 44

3.2 Recipe for the Callus induction media 1 49

3.3 Recipe for the Callus induction media 2 49

3.4 Recipe for the Callus induction media 3 50

3.5 Scale to score callus mass proliferation 50

3.6 Regeneration media 1 with MS Salt 51

3.7 Regeneration media 2 with MS Salt 52

3.8 Regeneration media 3 with MS Salt 52

4.1 ANOVA for No. of good proliferated callus 55

4.2 ANOVA for No. of low proliferated callus 58

4.3 ANOVA for No. of very good proliferated callus 60

4.4 ANOVA for No. of very low proliferated callus 62

2
 List Of Figures
Sr. No. Title Page.No.

3.1 Sterilization of seeds 42

3.2 Separation of each seed to become dry 43
3.3 Seeds were put in test tube for germination 43
3.4 Germination of seeds in test tubes 43
3.5 Plantlets were dried before dessication 45
3.6 Cutting of cotyledons to transfer on callus media 45
3.7 Callus induction on stem cutting 46
3.8 Callus induction on CM1 47
3.9 Callus induction on CM2 and CM3 48
4.3 (A) Graph of means of table 4.1 57
4.3 (B) Graph of means of table 4.2 59
4.3 (C) Graph of means of table 4.3 61
4.3 (D) Graph of means of table 4.4 63
4.4 (A) Type I Callus 64
4.4 (B) Type II Callus 65
4.5 (A) Callus Proliferation on CM1 67
4.5 (B) Callus Proliferation on CM1 68
4.5 (C) Callus Proliferation on CM3 69
4.6 (A) Regeneration from Callus 71
4.6 (B) Subculturing of regenerated callus 71
4.6 (C) Proliferation of regeneration phase in culture vessel 73
4.6 (D) Direct embryogenesis of green pods on regeneration 73
media

3
 Abstract

Shisham is one of the main timber sources of Pakistan and also grown on large scale
in other Asian countries. It is an important woody plant which has many commercial
benefits. For a better quality of the timber, it should be disease free and pest resistant with
few branches and the wider girth. In vitro propagation of woody plants has been less reported
worldwide but there are many fruit trees micropropagated by horticulturists. Tissue culturing
of explants result in the growth of a plant which is produced by only a tip cutting etc. For
high quality of the plantation, micropropagation is an indispensible aid which helps in the
multiplication of clones or callus. In this study the young plants grown from the mature
embryos and immature embryos on two different types of half MS media i.e with vitamins
and without vitamins and these young plants were used as explants. After germination these
explants were transferred to three different types of callus induction media i.e CM1(2,4,D
and Kn), CM2(BA and NAA),and CM3(2,4,D and NAA) for 15 days in dark. After this the
callus which had produced, shifted to three different types of regeneration media i.e RM1
(NAA and BA), RM2 (NAA and Kn), and RM3 (IAA and BA) for the induction of shoots.
For the selection of best media, propagation on different media was checked by using
different types of the callus and regeneration media.

4
CHAPTER 1

INTRODUCTION
Forests play fundamental role in our daily life by promptly providing us with shelter
and food. We being as humans depend on the forests for the air we breathe, for the wood
which we use and for our survival. Forests provide habitats to millions of animals and they
are also a support system for the numerous ecosystems. Forests have approximately 90% of
the species living on earth, both animals and the plants and make a diversity of forests
habitats for them. Forests like Amazon, provide watersheds and river systems and
recreational things. Plants and trees by the shedding of leaves help in stabilizing soil, manage
the cycle of water and by absorbing and storing carbon dioxide they play their role in
directing the climate of our Earth. Forests also enhance economically benefits such as they
attract tourists and tourism can enhance the earnings of native people of that areas.

Topographically Pakistan is a farming nation. As per monetary overview of Pakistan

2011-2012 woodlands in Pakistan covers the zone 4.80 % of complete area of the country
.The timberland zone of Pakistan is too little which isn't conquered the ideal proporation 20-
25% of the all out the region. This proportion is suggested by the FAO for the financial
improvement of the nation. In Pakistan the backwoods segment shares just 0.3 % every year.
The whole need of country is 3.5 million m3 of modern wood is given by timberland part
every year (Anon, 2006). Fuel wood, mechanical wood, paper, pulp and paper items, modern
and non-modern employments of wood are the most significant interest of our nation
Pakistan (Quraishi, 2005).

In Pakistan division of forestry has been as of now in emergencies because of

superfluous control and unfortunate behavior of woodland timber .On the decline in
backwoods and tree spread is a major issue or risk for normal agro biological systems and
condition. Numerous issue like contamination , soil and water are at a high rate. Numerous
untamed life species are losing their populace from the earth step by step (Rasheed, 2007).
The most plenteous and predominant ranch land tree species in Pakistan are Dalberia sisoo
42%, Acacia modesta 20%, Acacia nilotica 11%, Melia azadirachta 7%, Zizypus murtitiana

1
3.1% and some other minor species which are situated in unirrigated terrains of Pakistan
(Quraishi, 2004).

Shisham is a huge wide leaved tree species that has a non erect long stem (Singh,
2015). It is named after the Swedish researcher Nicholas Dalberg. It is found in tropical
zones of world for the most part in tropical districts of Australia , America and Asia
(Thothathan, 1987). Tallness changes next to important adaptable conditions inside the
inclines of Himalayas and a significant zone of Asian countries; Bangladesh, Pakistan ,
Kingdom of Nepal and India (Afzal et al, 2006; Bajwa and Mukhtar, 2006). 27 species are
accounted for in Indo-Pak subcontinent and 15 species are most regular in this region. The
most basic family of shisham is accounted for in Pakistan is Dalbergia sisoo Roxb.In 1866,
shisham was first time planted in Punjab in Changa Manga Plantation for coal creation on
bigger scale.

It is being an important woody plant has many commercial benefits and is mostly
found growing near the banks of rivers, streams, canals, with an elevation of 900m, but
naturally their range can reach upto 1500m. Shisham mostly grown on terrene soil. Shisham
is native to embankment of the Himalaya mountains of India, Nepal and Pakistan. Nepal is
considered for its centre of origin. It is also found near the sewage irrigated areas near
Khartoum in Sudan (Arya et al., 2013). The leaves of shisham are used for green manure and
fodder. The oil which is obtained from shisham is also used for heavy machinery as a source
of lubricant. The heartwood of shisham shows resistance to termites and its softwood is used
for the plywood. Along with the charcoal which is used for the different purposes; resins,
alkaloids, tannins and fibers are also obtained from it (Brijesh et al., 2006).

Dalbergia sisoo is commonly known as tahli, sheesham, sisu, and sisoo. Pakistan has
approximately 10,000 ha plantation of Dalbergia sisoo and it was first introduced in
Pakistan.in the mid of,,18th century. Dalbergia sisoo is important for its plantation in rural
and urban areas and is mostly planted because of its multiple uses and its fast growth.
Shisham is expansive leaved and spread deciduous tree specie which found in indo-Pak
subcontinent. It is known similarly as with different names in those locales, for example,
shisham, tahli, sisoo and Indian rosewood. The normally developing scope of shisham is for
the most part 900 to 1300 meters. The normal precipitation require for shisham development

2
is up to 2,000 millimeter and it can likewise endure in dry season conditions for most
extreme 3-4 months. (Ashraf, 2010).

Shisham is expansive leaved and spread deciduous tree specie which found in indo-
Pak subcontinent. It is known similarly as with different names in those locales, for example,
shisham, tahli, sisoo and Indian rosewood. Shisham is developed in timberland manor,
alongside roadsides, railroad lines, canals, water channels and agronomical fields. It is
generally develop alongside waterway banks in unadulterated sand and alluvial soil, it
additionally develop in saline soil. The normally developing scope of shisham is for the most
part 900 to 1300 meters. The normal precipitation require for shisham development is up to
2,000 millimeter and it can likewise endure in dry season conditions for most extreme 3-4
months. (Joshi et al., 2003).

It is not only well known for its use in timber but this tree also has its many medicinal
uses. It can be used as the expectorant, antipyretic, aphrodisiac, anthelmintic and
abortifacient (Hajare et al., 2001). Different species.of Dalbergia sisoo are ,important for its
production of timber and have value.for fragrance, decorative wood and have excess oils
which are aromatic (Bhattacharya et al., 2014).

Dalbergia sisoo also has its uses in the treatment of different diseases by traditional
methods. For example, its oil is used to treat scabies and buming. The leaves of Shisham are
used for the remedy of long hair and to remove dandruff (Lal et al., 2012). The extract of
Leaf of Shisham is used for the treatment of sore throat (Sultana et al., 2006). D.sisoo is also
used as folk medicine for the ailment of skin. The juice of leaves is also good for the nose
and eye disease (Shah et al., 2010).

Shisham is timber wood specie and is otherwise called a premium for rosewood
family. It has numerous various advantages, for example, it gives fuel wood, shade, shelter
and feed to animals. Due to these advantages it requires exceptional consideration against
various maladies and dry spell condition. Shisham is tall and erect tree specie, it regularly
accomplishes thirty meter of stature and 80cm in size under reasonable conditions. Bark is
thin, grey in shading , longitudinally wrinkled , shedding in fine strips , grows long tap root
framework at beginning periods of development and various sidelong ramifying roots .
Bloom shading varies from pale white to exhausting yellow and its cases pass on 1-4 seeds.

3
Timber gives an assortment of items , for example , nectar ,feed, fuel wood , tannins ,tar
,alkaloids and in like manner use for therapeutic reason. For money related purposes it is
additionally planted in atmospheres than locals, overall it is planted in China, Cuba, Brazil,
and so forth. In Cameroon nation individuals utilize their young leaves for the purpose of
eating. (Wang et al., 2011).

D.sisoo gives shade to various harvests like espresso and tea estate in tropical and
subtropical districts. These shades adoring yields are utilized for improving soil fruitfulness
under shisham. The wood powder and oil acquired from seed are utilized to fix distinctive
skin illnesses. Sisoo control soil disintegration through overwhelming development of
sprinters and suckers in various wastelands. Young shoots and leaves are utilized for grain
purposes, its leaves contain 2.7% to 24.1% unrefined protein and dry issue 32.46% which
gives support to creatures. It likewise have poison capacity which is utilized to shield crops
from various irritations and creepy crawlies. (Owra et al., 2009).

For building up shisham nursery, stump cutting are for the most utilized part. In
nursery, cuttings are developed for 6-8 months or for one year for the most part in beds and
after that move into field. The stumps which are utilized for build up the nursery are one
centi meter thick while those stumps whose distance across is more noteworthy than 2.5
centimeter are not appropriate for this reason. The cuttings of youthful tree are generally
utilized for nursery. For foundation of nursery occasional effect is additionally a significant
factor. It is seen that the planting which was done in August is exceptionally effective when
contrasted with planting was done in May and June. The reason is that May and june are dry
and hot while in August season of monsoon is begin. In 1981 Pain and Roy revealed that for
fruitful ranch, cuttings are doused for 30 seconds in various arrangements of development
controllers like IBA,and NAA individually.

Dalbergia sisoo is mostly known for its timber quality and with such multiple
products this specie of the rosewood genus should have greater consideration for the
applications of the agro forestry. For the forestry plantation, it is preferred that the plantlets
of the trees which yield timber should be of a good quality, disease and pest resistant with the
wide girth and few branches. Cloning the woody and superior material by the conventional

4
vegetative cutting method, which results in the uneven growth, as only cutting of tip gives
rise to the growing plant, and by branch cutting such trees can be produced which have the
characteristics as that of the branches. Micropropagation of the plants gives multiple clones
which give high quality of plantation. Tissue culture of D. sisoo is done to meet the quality
and quantity demands of the plants (Arya et al., 2013).

Two diseases namely wilt and dieback are accounted for to cause Shisham declined in
Pakistan and neighboring nations. Shisham is most normal sickness in boggy a waterlogged
regions , where the manor is single or reduced (Baksha and Basak, 2000) . In 1998 Shisham
plant malady appeared as most noticeably terrible structure in central immersed zones of
geological locale. In Pakistan and neighboring countries this disease has made tremendous
scale mortality of Shisham trees with the strange condition of solemn (Naz et al., 2002).
Shisham has declined due to dieback disease which causes threat to this specie. The
maximum mortality rate related to this specie was observed along the banks of canals which
is about 40% along with the road. The plants which grow in the well managed area and on
the agriculture land were reported to be least affected by this disease and have less mortality
rate.

Normal most sicknesses in Dalbergia sisoo are wilt and dieback. In wilting condition
the underlying foundations of plants is influenced by growth and plant show indications like
withering of units, leaves and twigs (Ali et al., 2012). In dieback condition the affected tress
bring about diminishing of crown in view of loss of leaves, crown shading transform into
yellow shading, the shoot tips and twigs get to be plainly dry. The illness affected tree
develop close to the establishment and red-chestnut cylinder formed structure cambium gets
to be plainly dry that spreads from underneath the establishment to 1.5 meters over the base
dimension. Underneath the bark, diminish lines are delivered and spread over the tissues
influencing the entire wood. The roots which are contaminated can't recuperate and the tree is
accounted to be dead. (Bajwa and Mukhtar, 2006).

Shisham die back sickness is more dangerous than wilt. The yield misfortune because
of dieback disease is over 21%. The causing agents of dieback disease are generally
Rhizoctonia solani, Curvularia lunata and Fusarium solani. The agents cause decreasing of
crown and leaves, shoot tips and twigs to be dry and they also dry the primary stem of

5
shisham tree. Shisham declined has brought about substantial monetary misfortunes and
furthermore decreased the use of shisham wood. Breeding methods which are conventional
and the morbid involvement resuscitate the Shisham however they helped Shisham in its
survival.

Biotechnological mediations are yet to be genuinely connected for its improvement.

The present issue is one of such movement which will prepare to apply biotechnology for
Shisham improvement. In vitro regeneration and tissue culture is used for manipulation of the
genome of tree. Several reports have come out which are based on the tissue culture of
D.sisoo using the cotyledon, hypocotyl, nodal explants. Shisham is being attacked by two
different diseases wilt and dieback and the older trees are greatly affected by these diseases
(Bajwa et al., 2003).

Breeding.methods which are traditional are difficult to some extent and consume
time. So, not only for the varietal improvement some methods should also develop for the
propagation at the large scale. With the advanced methods in the plant propagation, the
micropropagation and the culture which is in vitro has enactment as.method for the
production,of plants at a large scale and are much considered better for the production of
seedlings which have better quality in a short period of time. Propagating plants by this
method increased the genetic, discrepancy in a relatively much shorter time. This technique
is being used for propagating forest trees.

Presently days this system is being utilized for the improvement of many timberland
trees (Gyvess et al., 2011; Rathore et al., 2007) particularly by somaclonal variety, in vitro
incited mutagenesis and hereditary change through callus culture. The point of this
examination was to institutionalize in vitro micropropagation framework for D. sissoo by
changing the sythesis of plant development controllers (Auxins and cytokinins) in the
medium during the various phases of the way of germination and regeneration. Principle
criteria used to choose best micropropagation conditions were improvement of increase rates,
shoot quality and establishing effectiveness.

Micropropagation of plants is a priceless guide in the duplication of world class

clones for advancement of top notch estates anad is to expand profitability both
quantitatively and subjectively just as to meet the preservation perspective which is the need

6
of the hour. Conventional rearing techniques are excessively troublesome and tedious. Along
these lines, there is a critical need to present some propelled strategies for mass scale spread
as well as for varietal improvement. Among cutting edge strategies for plant proliferation, in
vitro culture or micropropagation has been built up as a technique for mass scale spread of
plants and truly encouraging for the creation of good quality seedlings in generally shorter
range of time. This strategy likewise gives expanded hereditary inconstancy in moderately
shorter time without applying an advanced innovation.

The proposed study will standardize the in vitro micropropagation method by

amending the constitution .of different.growth regulators of plants in the media
during.various,stages of culture. The best composition will use to select the best
micropropagation media for the multiplication quality of shoot and the efficiency of rooting.
Distinctive customary techniques are utilized to control and create malady free plant like
propagation of seed of D.sisoo however the outcomes was bad. Along these lines, by the
utilization of tissue culture technique infection free plants with better characters can be
created.

7
CHAPTER 2

REVIEW OF LITERATURE
Rehman et al. (2012) developed the protocol.considering the plantlet.regeneration and
for the formation of callus by culturing the mature embryos, seeds which are immature and
using cotyledons, on the (MS) medium which was supplemented with the growth regulators
of plants. Three types of media which are callus inducing were used with various
concentration of hormones and the media which produce better callus contained 2.71uM 2,4-
D (dichlorophenoxy acetic acid) and .0.93 uM kn and shoot was also regenerated on three
types of media and the best treatment was 1.4uM NAA and 8.88uM BA and better rooting
was done on the MS half media with the 7.35 uM indole-3-butyric acid.

Arya et al. (2013) developed the micro propagation in clones of Dalbergia sisoo
which were collected from different regions based on their phenotypic characters. The clones
established and were maintained with rapid proliferation of the axillary bud proliferation.
Nodal explants sterilized with 0.1% concentration of mercuric chloride solution for 15mins.
For the collection of explants the effect of the season was deemed. In clone no 9, the
maximum frequency response at optimum requirement was 92.35% and in two clones the
response was more than 89% and 91%. Different multiplication rate was observed by using
BAP at 2.5um in the in vitro shoot multiplication. IBA supplemented medium was used for
in vitro root induction. For the induction of roots by in vitro IBA supplemented medium is
used.

Nayak et al. (2007) used the cotyledonary nodes of Aegle marmoles to develop the
planlet regeneration. The cotyledonary nodes of 1 month old in vitro seedlings were cultured
on the MS medium which contain kinetin ranging from 0uM to 9 uM, and the IAA ranging
from 0uM to more than 1.5uM and BA ranging from 0uM to 8uM. The best regeneration was
obtained on media which contain 6.6 uM BA and 1.14 uM IAA. Cultures which were
maintained on the kinetin media didn’t show good response. The plantlets which show
regeneration of shoots were transferred to rooting the media for the induction of roots on half
MS media which contain NAA, IBA, and IAA. Rooting was best observed in the media

8
which contain the best required concentration IBA. The plantlets which showed roots were
shifted to field with survival rate of 80%.

Joshi et al. (2003) examined the results of clonal propagation of Dalbergia sisoo on
different nutrient media. Nodal explants were taken from the two cotyledons which were
cultured on the two nutrient media MS and B5 and were formed from the both media within
6 to 8 days with different combinations of the media which were supplemented with BAP
with the combination of NAA or IAA. NAA gives the maximum number of shoots. MS
media gives the best result for the proliferation of shoots in comparison with the B5 media.
Within 18 days IBA (1.0 mg/l) in half MS gave the best results for the number of roots. The
plantlets which were produced acculturate and were shifted to pots and rate of survival was
achieved upto 70%.

Ali et al. (2012) made attempts for evaluating the best culture conditions for the
micro propagation of nodal meristem of Dalbergia sissoo. Within 10-12 days the media
which contain 0.25mg/l NAA +.1.0 mg/l BAP in MS media shows the best response of shoot
formation and after 25 days average, length of shoot was observed upto.2.4cm. After the
inoculation of 4 weeks, the average of .length of shoot was 1.78 cm and maximum shoots
were observed by the MS media with the combination of the O.25mg/l Kinetin + 1.5mg/l
BAP. Shoots which were well developed, transferred to the root media which was MS media
and boosted with the 1mg/l of the indole butyric acid (IBA) and after 24 days of inoculation
3 to 4 roots were developed per plant with average.length of 1.8 cm. Different conditions of
temperature which had provided ranged from 23 to 30c to culture but the photoperiod at 26c
gave the best optimum results.

Bari et al. (2008) studied complete and efficient protocol for callus induction, plantlet
regeneration from internodal and nodal segments which are in vivo and the tips of shoots of
Dalbergia sisoo. The MS media which was constituted with 0.5mg/l amount of NAA and
2mg/l amount of BAP gave the highest percentage of induction of callus from the nodal
segment. By the use of this combination, friable green callus and the highest weight callus
produced was fresh. For the internode segment the maximum shoots were obtained from MS
media which was provided with the 0.5 mg/l amount of IAA and 1.5 mg/l amount of BAP.
The combination of 2mg/l amount of NAA and 1mg/l amount of IBA gave the maximum

9
number of roots. Theplantlets which were well rooted, acclimatized were successfully
transferred to soil and the chances of survival of the plantlets were about to 60%.

Singh et al. (2002) generated buds of shoots were from cotyledons which were semi-
mature on the MS medium which were supplemented with 0.26 uM naphthalene acetic acid
(NAA) and the 4.44 uM 6-benzyladenine.The buds which prolonged into the shoots
transferred to the media similar to that which contained the half strength macro-nutrients.
Mature cotyledons were induced the adventitious shoot bud formation. The mature
cotyledons as compared to the semi mature cotyledons showed the differentiation of shoot
bud on the MS medium which contained the 22uM BA without the NAA. Regeneration of
shoot bud was improved approximately upto 6 fold by preculturing the mature cotyledons in
the liquid MS media for duration of 48h which contained 2.2 uM. The regenerated shoots
which were taken by the mature and the semi-mature cotyledons were transferred for roots
development on the half MS medium which contained the 4.92 uM and 1.23 uM indole-
butyric acid (IBA) respectively. Seedlings maintained and were shifted in soil.

Anis et al. (2003) used the cultures which were primary of the Morus alba L. on the
full MS media which were constituted .with the BAP along with kinetin and observed higher
amount of germination from the nodal which is 80%.and from the explants of shoot
differentiation and tip it was 70%. The MS medium supplemented with the BAP which was
taken as 2mg/l) and the amount of NAA which was taken as 0.2mg/l showed rapidly
multiplication of the proliferated shoots by the cultures of the shoot tips and the nodal
explants which were produced in vitro. This combination was considered to be best for the
multiplication of formation of shoots. With culturing both kinds of explants on the MS media
which was fortified with the aspargine, BAP, glutamine and NAA. This fortified media
improved the extention of the shoots and proliferation of axillary buds of the shoots which
were grown in vitro. When shoot cultures were transferred to the MS medium was
constituted with the NAA, about 80% rootings was observed. Plants were shifted to soil
which had well developed roots with the survival frequency of the 70%.

Islam et al. (2008) did in vitro propagation and checked the effect of regulators which
were related to growth on the nodal explants of four species of Yam and for favourable
production of microtubers the lower amount of..Kn (5mg/l) with the IAA along with the

10
sucrose was proved to be the best combination. But the existence of the BAP badly affected
the proliferation of the mictrotubers.

Zhou et al. (1984) contrasted the callus-promoting activity of the most frequently
known and some rarely tried auxins with that of 2,4-dichlorophenoxyacetic acid (2,4-D) for
the excised spring and winter wheat embryo in vitro culture i.e. Chinese and Chinese Spring.
Fredrick. Different auxins showed commonly distinct operations in a concentration range
from 1 to 50 μM. The two wheat cultivars also reacted to the auxins differently. Propionic
acid dicamba, picloram, butyric acid, 2, 4, 5 trichlorophenoxyacetic acid, and 2, 4, 5-
trichlorophenoxypropionic acid were used as the basis for comparison. All auxins tested
promoted plant development; 2-methyl-4-chlorophenoxypropionic acid was most efficient
for Chinese Spring, whereas picloram was most efficient for Fredrick.

Adebe et al. (2008) utilized the youthful embryos as an explant for recovery of maize.
The immature embryos were carefully constrained for a reasonable span to culture. Mature
zygotic embryos harvested from dry plants, on the other hand, are omnipresent. However, for
tissue culture job, usually mature embryos and particularly tropical maize genotypes were
regarded the most recalcitrant. The maximum average callus induction recorded using LS
basal salts and B5 vitamins supplemented with 3 mg/l 2,4-D alone was 90 percent and 52.5
percent when same level of 2,4-D was combined with Kinetin and the regenerated calli were
maintained on the media consisting of LS basal salts and B5 vitamins supplemented with 2
mg/l of 2,4-D. The frequency of regenerable calli recorded was 21.14 percent. The amount of
shoots caused on single divided seed regenerated per callus varied from 1-5.

Abdellatef et al. (2008) launched the callus culture on B5 basal media from hypocotyl
explants of Barac B-67 staple cotton cultivar. Different kinds and levels of growth regulators
were evaluated to achieve the finest formation of calluses.In this research, four auxin kinds
were used in conjunction with NAA, IAA, NAA, IBA, 2,4-D at five concentrations and two
cytokinines, BA and Kinetin at four levels. The highest callus growth index frequencies 8.13
and 8.0 were observed on hypoctyle explants grown on basal B5 supplemented with 1mg/l
NAA in conjunction with 0.1mg/l Kin or BA, respectively. The medium containing Kin led
in the creation of compact callus, resulting in big amounts of roots. On the basal medium of

11
B5 there was no callus formation. The caused callus on the 2, 4-D B5 medium was brown in
colour and of low quality relative to that generated on NAA-containing B5 media.

Michel et al. (2008) selected ten genotypes of cotton for tissue cultivation. The
initiation of callus was dependent on genotype, and R405-2000 has the best response to
callogenesis. The proportion of callus induction and dry weight of callus varied from three
media, but MS was the finest callogenesis medium. It seemed that callus induction from
hypocotyl was much simpler than cotyledon or root explants. Cotton induction callus varied
with hormone regimes. In effect, the callus initiation was encouraged by a correct mixture of
2,4-dichlorophenoxyacetic acid and kinetin. Glucose was the finest sugar to encourage callus
manufacturing.

Rathore et al. (1991) generated multiple shoots by in vitro from Tecomella undulata
nodal shoot sections chosen for quality of wood and rapid development. Approximately 8–10
shoots were obtained within 2–3 weeks from a nodal explant on the medium of Murashige
and Skoog (MS) supplemented with IAA and BAP at 31°C during a photoperiod of 12 hours
/ day. The in vitro shoots could be subcultured on a new MS medium containing IAA and
BAP to further multiply. These shoots could multiply without loss of vigor for 12 months. It
was discovered necessary to subculture the shoots to the new medium within 3–4 weeks. MS
fluid medium containing IBA for 48 h was rooted in isolated shoots. Then transfer to the
hormone-free MS medium of half intensity. An original 5–7 day dark period favored root
induction. Thus acquired plantlets have been hardened and transported to pots.

Sharma et al. (1988) evaluated that when grown on Murashige and Skoog (MS)
media containing BAP + IAA / IBA / NAA, the hypocotyl explants of Dalbergia sissoo
which were long about one cm generated the shoot buds. Shoot buds were also induced on
the MS medium with BAP + IAA from isolated hypocotyl callus. The isolated shoots may be
rooted in the combination of MS medium + IBA, NAA or IAA, or IBA with combination
of NAA.

Assem et al. (2014) optimized the tissue culture conditions for Sorghum bicolor
through somatic embryogenesis from immature embryos of 10 sorghum varieties were
assessed on distinct culture media for their renewal potential which contain adequate
combinations of growth regulators. The ideal response of embryogenic callus induction was

12
achieved from Murashige and Skoog (MS) cultured explants supplemented by proline and
2,4-dichlorophenoxyacetic acid (2,4-D). Some varities regenerate while some didn’t show
the procedure of regeneration. Regenerated shoots developed into mature plants that were
normal. The 2,4-D auxin was critical for embryogenic calli induction. Adding cytokinin
(kinetin), however, adversely impacted embryogenic callus formation. On the other side, the
induction of the plant was more affected by the addition of thidiazurone (TDZ), thidiazurone
(TDZ) and indole-3-acettic acid (IAA).

Pattnaik et al. (2000) identified effective protocol for plant regeneration from cell-
suspension cultures of Dalbergia sisoo. Callus induction on a medium containing naphthalene
acetic acid (NAA) was superior of the two distinct auxins tested. The proportion of callus
induction improved significantly when BA was added to NAA. Hypocotyl sections were
most responsive of the three distinct explants assessed for callus induction. On a medium
supplemented with a combination of BA and NAA, shooting organogenesis from callus
cultures was higher than on BA alone. On a medium comprising 13.3μM BA 2.7μM NAA,
71% of crops exhibited shoot-bud differentiation. The shoots which regenerated were rooted
on an indole-3-propionic acid, indole-3-butyric acid, and indole-3-acetic acid and half-
strength MS medium. Plantlets in the soil have been acclimatized and developed.

Norma et al. (1987) introduced the methods of tissue cultivation for cotton
enhancement. Preliminary screening for embryogenic potential of eight strains of Gossypium
hirsutum L resulted in somatic embryos being produced in all strains of cell suspension
culture. For use in developing a model regeneration scheme for G. hirsutum Coker 312 was
chosen. Calli were produced from hypocotyl tissues of old seedlings which were of 3 days.
After six weeks of culture, globular embryos were present. Calli were subcultured in a
growth regulator-free medium to liquid suspension. Suspensions were sieved after three to
four weeks to collect embryos from the globular and heart stage. When placed on a semi-
solid medium, collected embryos created further. Mature embryos were put on medium-
saturated sterile vermiculite to cause germination and plantlet development. When the roots
and two true leaves were developed, the plantlets were potted and hardened in peat and sand.

Finer et al. (1984) cut stem and petiole parts from a diploid cotton species and put on
a callus induction medium. Explants with the cut surface either browned or created a slowly

13
growing red callus that could not be subcultured in contact with the medium. Explants with
an epidermis in contact with the medium generated a quickly increasing, friable, green callus
that could be subcultured biweekly and kept for 12 months. Two weeks after the callus was
transferred to a liquid medium containing auxin, 2, 4-dichlorophenoxyacetic acid, globular
and torpedo embryos were obtained. The embryos underwent further development after
subculture to an auxin-free medium, but plantlets were not obtained

Trolinder et al. (1988) put the cotton calli produced from seedling hypocotyl tissue in
liquid suspension and kept in the hormone-free MS medium by serial subculture
.Suspensions have been sieved and globular embryos have been gathered, washed,
resuspended in basal media and plated on multiple semi-solid media. Proliferation and
maturation of long-term embryos were best on average MS plus KNO3. From 10.6 percent of
embryos, plants were recovered. When embryos of about 5 mm were placed on the medium,
within six weeks, 30% of embryos formed plants. Smaller embryos required a longer
development period on the vermiculite and adding 0.1 mg / l GA3 to the fresh medium.
Plants with an expansive root system and two true leaves were separated from sterile culture
and potted into either peat and sand, or vermiculite, one - to-one. Eighty percent of the
regenerants were effectively hardened during a two-week period.

Maddock et al. (1983) grew immature embryos and wheat inflorescences on agar
media. The presence of auxin 2, 4-D caused various shoots to be formed in a percentage of
cultures. The shoots evolved out of structured, embryoid-like structures in some instances.
Comparison was made between the morphogenetic capacities of 25 different spring and
winter wheat cultivars, and clear differences between genotypes were found. Shoots were
cultivated individually and the resulting crops in the glasshouse were cultivated to maturity.

Lazar et al. (1988) identified the yield of haploid plants from other cultures can be
significantly influenced by cultural media and environmental factors. Their goals were to
identify a combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid
(IAA) concentrations that produce the maximum number of haploid plants, and to evaluate
the effects of induction medium duration on calli induction, plant regeneration, and green
plant production from other spring wheat crops. The use of response functions to estimate the

14
maximum effectiveness of 2,4-D and IAA combination implied that higher 2,4-D levels
should be investigated in the induction medium and that the optimum hormone combination
differs for regeneration of plant and the percentage of green plants. For plant regeneration
and green plant proportion, significant impacts of length on the callus induction medium
were noted.

Huang et al. (2004) created an effective maize regeneration method by using mature
embryos. Embryos were removed from mature seeds with surface sterilization and cut into
halves. They were used as explants to trigger callus supplemented by 2,4-D on the induction
medium. Primary calli induction frequency was over 90 percent for all tested inbred lines.
Embryogenic calli were formed after subculturing for every two weeks. On the regeneration
medium, the embryogenic callus readily formed plantlets supplemented by 0.5 mg/l BA. To
create good roots, the regenerated plantlets were transmitted to the semi-strength medium
Murashige and Skoog supplemented with 0.6 mg/l indole-3-butyric acid. The regenerated
plantlets succeeded in transferring and setting seed to the soil

Sujay et al. (2004) investigated five elite inbred lines of maize using 14-day immature
embryos as explants to induce and regenerate callus. Explants cultivated on the medium of
Murashige and Skoog (MS) supplemented with 2,4-dichlorophenoxyacetic acid at 1 mg/l
demonstrated the greatest callusing frequency. Explants cultivated on the N6 medium gave
the largest organogenic callus frequency among all the media tested. N6 added with 2 mg l−1
Dicamba produced the greatest organogenic callus frequency. Among the five genotypes
tested, the best callus was given by CM 124, CM 125, and CM 300. When shoots were
cultivated on MS medium supplemented with 2 mg/l naphathalene acetic acid, the largest
frequency of root formation was noted. Regenerated plant percentage ranged from 54 to 66.

Jiang et al. (2010) evaluated the effects of three different dicamba concentrations and
two different types of sugar on callus induction and plant regeneration from mature embryo
to establish a highly efficient plant regeneration system for wheat genetic transformation on
basal medium L3. The findings showed that genotypes, sugar types and dicamba levels
considerably affected the effectiveness of mature embryo culture. 4 mg/l dicamba proved the
best efficient to induce embryogenic callus and also provided the highest percentage of
regenerated plants in both cultivars. Replacing maltose with sucrose considerably enhanced

15
the effectiveness of plant regeneration in both cultivars. These findings will facilitate job
with elite wheat on genetic transformation.

Maria et al. (2002) undertaken this research to improve the induction of callus and
plant regeneration from cultivar' Bobwhite ' wheat mature embryos. Effects of four auxins
2,4-D, dicamba, picloram, and 2-MCPP and effects of maltose vs. sucrose were evaluated
under filter sterilized and autoclaved conditions. All auxin combinations with the exception
of 2 MCPP led in callus induction. The sugar type impact depended on the type of auxin that
was used. Substitution of sucrose with maltose increased the capacity of callus to regenerate
from embryos grown on media containing 2,4-D and picloram, but caused an inverse impact
on dicamba-containing media. Picloram considerably increased callus development. Dicamba
(18μM) in relation to 2.4-D resulted in a double increase in the number of regenerated plants
per embryo and reduced the time required for plant regeneration by 3–4 wk.

Bronsema et al. (2001) optimized seed sterilization procedures of distinct 55 cotton

rows. In the open field seed collection of cotton lines, the germination rate was very small.
Different sterilizing agents were used for sterilization.Germination rate was very high and the
rate of contamination with hydrogen peroxide was very small. Successful protocol of all
genotypes, including autoclaved water wash, ethanol rinse and hydrogen peroxide
sterilization, was created for 7h.

Ozgen et al. (1998) grew genotypes by in vitro method to create an effective

technique of callus formation and crop regeneration from mature embryo culture, and to
compare the reactions of both embryo cultures., immature and mature embryos of 12
prevalent winter wheat (Triticum aestivum). After fifteen days , immature embryos were
dissected aseptically from plants and put upwards with the scutellum on a strong agar
medium containing MS and 2,4 D. For callus induction, the seeds with moved embryos were
put furrow down into plates comprising 2, 4-D. On 2, 4-D-free MS medium, the calli and
regenerated plants developed were maintained. Regenerated plants from both cultures of
embryos have been vernalized and cultivated in soil to maturity.

Yu et al. (2019) evaluated the effects on callus physiology and its growth of distinct
colored light. During embryogenic callus developments, expression and induction stages
were extremely influenced by various light characteristics. Red light had a positive effect on

16
the embryogenic callus induction stage, but the differentiation period was lowered to 30 days
compared to white light. Red light also preserved the equilibrium of various enzymes for
callus induction peroxidase, dismutase and catalase. In embryogenic calli, the levels of auxin
were considerably increased under red light medicines. Using red light treatments, highly
efficient and relevant protocol for callus induction was created.

Wu et al. (2004) developed an effective protocol for 10 recalcitrant cotton genotypes

for embryogenesis and plant regeneration. Optimum kinetin and IBA concentrations were
used for callus induction on the MS medium. For 86.7% callus distinction into embryos, the
MSB2 medium supplemented with double quantity of KNO3 was used. For 38.3%
transformation of embryos into crops in 8 weeks, MSB3 medium supplemented with
glutamine and arginine was used. Commercially significant protocol for recalcitrant cotton
cultivars has been created.

Michel et al. (2008) selected ten genotypes of cotton for tissue cultivation. The
initiation of callus was dependent on genotype, and R405-2000 has the best response to
callogenesis. The proportion of callus induction and dry weight of callus varied from three
media, but MS was the finest callogenesis medium. In effect, the callus initiation was
encouraged by a correct mixture of 2,4-dichlorophenoxyacetic acid and kinetin. Glucose was
the finest sugar to encourage callus manufacturing. The optimum callus induction glucose
concentration was 40g/L. MS medium with 0.1mg/l 2,4-D, 0.5 mg/l KIN and 4 percent
glucose was the finest medium for callus proliferation. An effective protocol has been created
to produce high-frequency cotton callus.

Sakhanokho et al. (2001) created protocol for the production of crops from somatic
embryos in various cotton species. Cotyledons and hypocotyl sections were grown in
addition to NAA and kinetin on callus induction MS media. Embryogenic calli were
separated from the media and transferred with double quantity of KNO3 to embryo initiation
MS media. Calli were gathered for embryo maturation and moved to the next media. Somatic
embryos on MS media have been further created into plantlets. It is a very simple and helpful
method to enhance germination in various cotton species.

Chowdhury 2011 explored the impact of cytokinins on the proliferation of calluses

from cotyledons and the growth of plantlets in cotton. The frequency of callus induction was

17
noted at distinct levels on MS medium enriched with a variety of cytokinins. The proportion
of callus formation, percentage of shooting growing calli and amount of shoots or calli were
improved with the rise in cytokinin concentration. BA showed the greatest output among the
three distinct cytokinins studied. On MS media supplemented with 1 mg/l Kinetin, the
highest amount of shoots was observed. The rooting media consisting of MS medium, 0.6
percent agar, sucrose and NAA fortified induced root growth at the largest proportion with
maximum amount of roots per cut and root length per crop. In natural circumstances, the
plantlets were acclimatized.

Chen et al. (2008) improved callus induction and regeneration of plants using
hypocotyl explants for four distinct cultivars of cotton. By altering the kinetin concentration
with 2, 4-D, different hormonal combinations were screened for callus induction. Using
double quantity of KNO3 for hypocotyl sections has enhanced callus induction. For calli
induction and somatic embryos, high kinetin concentration and lower 2, 4-D concentration
reacted best. Plantlets were generated under dehydration circumstances from somatic
embryos. Finally, for cotton cultivars, the best one regeneration protocol has been created.

Tripathy et al. (2002) used ten distinct cotton varieties for callus induction and plant
regeneration For various variants, leaf, stem, hypocotyl, cotyledon and leaves have been
used as explants. In various combinations for callus induction and plant regeneration, distinct
hormones TDZ, IBA, 2PI, Kinetin, NAA were used. MS media, including IBA and IAA, was
the best among various basal media for plant regeneration. NA920 cultivar reacted best with
a regeneration frequency of 8.2 percent for callus induction and plant regeneration.

Mahin et al. (2016) used hypocotyl as an explant of various Iranian verities i.e.
Hashem abad, Kerman, Termez and Sepid for callus induction and compared it to Coker 312
cotton. For callus induction, the MS medium comprising B5 vitamins, 2, 4-D and kinetin was
used. The following media MSB1, MSB2, MSB3, and MSB5 used different hormone levels.
Nearly 100 percent calli induction was found in Coker 312 using MSB1, MSB2 and MSB4
media, and only 46.66 percent calli were caused by Hashem abad culture in MSB3 medium.
Compared to Termez, Sepid and Hashem abad, the proportion of Coker 312 embryogenesis
was considerably greater. In the Kerman genotype, there was no embryogenesis.

18
 Ikram (2005) chose Coker-312 cotton variety to observe the impact on callus
proliferation of various chemical compounds in callus induction media. The effect of
nitrogen concentrated primarily on cell growth, callus induction and the manufacturing of
anthocyanin. Different hormonal combinations of 2, 4-D and kinetin were used to produce
embryonic callus. On MS medium, which contained ammonium nitrate and potassium
nitrate, embryonic callus was then cultivated. Potassium nitrate has been noted to have a
positive effect on the proliferation of the callus, but ammonium nitrate has a negative effect.

Behera et al. (2008) created a regeneration protocol by using cotyledonary nodes for
Gmelina arborea Roxb,. Multiple shoots were caused either alone or in combination with
either IAA or NAA on the medium of Murashige and Skoog (MS). Higher amount of shoots
were generated by cotyledonary nodes cultivated on medium with BA and IAA. Cultures
supplemented in TDZ showed bad reaction. In vitro regenerated shoots were grown either
alone or in conjunction with 2, 3, 5-triiodobenzoic acid onto a root induction medium
consisting of half-strength MS supplemented with IAA or indole-3-butyric acid. Rooting was
best complemented by IBA and TIBA in the medium. Rooted plantlets were acclimatized and
transmitted to the field at 70 survival rates.

Thirunavoukkarasu et al. (2007) accomplished shoot multiplication from BA and

IAA supplemented cotyledon and hypocotyl sections of in vitro germinated Enterolobium
cyxclocarpum seedlings on Murashige and Skoog's medium. Among the distinct levels and
combinations tested, BA in conjunction with IAA triggered the best response in terms of
cotyledon explant shoot bud formation. Such organ genic effect was shown by a maximum of
80 percent of the hypocotyl explants when grown on MS medium supplemented with 1.5
mg/l BA and 0.2 mg/l IAA. In addition to either IAA or IBA, elongated shoots could be
rooted on the half-strength MS basal medium. Explants of the cotyledonary node showed bad
reaction.

Saito et al. (2012) created a synthetic seed formation technique by encapsulating in

aseptic circumstances axillary buds from in vitro cultivated plantlets of two species of fast-
growing tropical tree, Gmelina arborea and Peronema canescens. MS medium comprising
plant development regulators benzylaminopurine, α-naphthylacetic acid, 3-indolebuthylic
acid and distinct levels of sucrose were used as an encapsulation medium in conjunction with

19
4% sodium alginate. Elongation in shoot G. Arborea has been promoted in the encapsulation
medium at reduced levels of sucrose, whereas P. canescens has been promoted at greater
levels of sucrose. By transplanting the elongated shoots into vermiculite, plants of these
species were created within 1 month.

Shilpa et al. (2014) relied the concentration and mixture of PGRs to form various
shoots. 6-benzylaminopurine alone provided the greatest effectiveness in regeneration among
all PGRs. Likewise, Indole-3-butyric acid was the most effective PGR in the microshoots to
induce root formation. The regeneration effectiveness was also influenced by media
composition and carbon source. In this order, MS medium demonstrated to be the finest
regeneration medium followed by B5, Schenk and Hilderbrandt medium and woody plant
medium. Based on this research, using glucose for maximum regeneration was recommended
for effectiveness instead of sucrose in the MS medium.

Ijaz et al (2016) discovered at the commercial stage, plant micropropagation has

become an imperative approach that delivers big numbers of crops in less time and space.
Micropropagation in woody trees is more tedious and complicated than other crop crops.
Eight micropropagation media with different combinations of benzylaminopurine and indole
acetic acid have been researched for this purpose. As explants, auxiliary and epicormic
shoots of Dalbergia sissoo were used with at least one nodal bud. MPMS5 is the highest
blend of mediums that gave the highest amount of shots followed byMPMS7.

Khan et al. (2002) developed a protocol for Bixa orellana L seed callus plantlet
regeneration. Seeds showed elevated proportion of callus induction and elevated yield of
black friable callus on the Murashige and Skoog medium comprising naphthaleneacetic acid
and N6-benzyladenine within 6 wks of dark. Increased myo-inositol and ascorbic acid to the
crop medium enhanced the frequency and development of callus induction. Shoots extended
to 4 cm within 4 week of transferring to MS without growth regulators. Shoots were rooted
using IBA on half-strength MS medium. Approximately 85% of these crops were created
after 3 wks of hardening in pots comprising pure garden soil and organic manure.

Pinto et al. (2002) acquired somatic embryos by a 60-yr-old Quercus suber L tree. On
the Murashige and Skoog medium, leaf explants were grown with sucrose, gelrite, 5.8-
adjusted pH and various combinations of growth regulators. During the first 3 weeks, callus

20
induction took place in the dark. After 3 months, without growth regulators, calluses showing
embryogenic structures were transmitted to the same medium. Only calluses caused on the
E3 medium supplemented with 2,4-dichlorophenoxyacetic acid and zeatin showed somatic
embryogenesis. On average, 10% of somatic embryos germinated and 40% of these
germinated embryos were transformed into crops. Without growth regulators, plants were
elongated on the same medium and acclimatized to greenhouse circumstances.

Bueno et al. (1992) handled cork oak with various 2, 4-D levels of zygotic embryos,
endosperm and ovules to induce somatic embryos. During the season of embryo growth,
plant material was gathered from June to September. The most reactive original explant was
the immature embryos. After the start of the 2,4-D therapy, callus and somatic embryos
produced after few weeks. Different growth regulators and cdesiccation treatments have been
screened for embryo development studies. The best treatment to break dormancy was cold
storage of somatic embryos matured in vitro at 50C.

Cuenca et al. (1999) induced the somatic embryogenesis by seedlings of internodal

and leaf segments of pedunculate oak which were used as an explant source. Auxin therapy
affected embryogenic reaction that happened only in explants originally grown on media
comprising naphthaleneacetic acid and various levels of benzyladenine. After 6 weeks of
culture on the induction medium, the explants were transmitted to the medium supplemented
with BA and NAA , and 4 weeks later subcultured into a growth-free medium. Different
embryogenic lines were created and retained in a multiplication medium comprising BA plus
NAA by repetitive embryogenesis.

Fernandez et al. (1995) acquired somatic embryogenesis from young Quercus suber L
seedlings in leaf cultures. A two-stage method was needed to start the process by adding
benzyladenine and naphthaleneacetic acid first at elevated levels and then at low levels.
Somatic embryos emerged when the explants were subsequently put on plant growth
regulators that were mixed in the medium. The embryogenic lines stayed productive on the
medium without growth regulators through secondary embryogenesis. Under these
conditions, 15% of embryos had coordinated root and shoot development and 35% had either
shoots or mostly roots. These percentages were greater than the percentages of darkly
matured embryos.

21
 Manzarena et al. (1993) created embryogenic callus on immature zygotic embryo
hypocotyl under the impact of 2, 4-dichlorophenoxyacetic acid (2, 4-D) in both the liquid and
agar media. Chilling but not desiccation treatments caused germination. Epicotyl dormancy
has been overcome by putting the embryos on the medium with 6-benzyl adenine with
elongated radicles. Normally advanced plantlets have been transmitted to the ground and
acclimatized in a greenhouse.

Gautum et al. (1993) originated the callus on Nitsch or Murashige and Skoog's
medium supplemented with growth regulators. A greater proportion of crops generated
Nitsch medium callus comprising 10 μM of indole-3-acetic acid and 1 μM of 6-
benzyladenine. In 25 percent of the crops on Murashige and Skoog's medium, 10 μM α-
naphthaleneacetic acid and 1 μM kinetin, green nodular structures and prominent roots
created after 13–15 weeks. In this anther-derived callus, multiple shoots were caused when
subcultured with benzyladenine and α-naphthaleneactic acid together with
polyvinylpyrrolidone on the medium of Murashige and Skoog. After subculturing on the
medium supplemented with indole-3-butyric acid and polyvinylpyrrolidone of Murashige
and Skoog, the excised shoots created roots, thus creating full plantlets.

Rout et al. (1993) regenerated plant through somatic embryogenesis was

accomplished through callus obtained from Acacia catechu wild's immature cotyledons.
Adding 0.9–3.5 mM of L-proline to the medium influenced somatic embryo growth as well
as encouraging secondary somatic embryogenesis. The light-green somatic embryos
germinated with 2% sucrose on a half-strength MS medium. Somatic embryos germinated in
greenhouse acclimatized plantlets and then moved to the ground.

Yew et al. (2011) use three Morus (mulberry) species and these caused callus and
adventitious roots. The Sugye roots had the largest rutin concentrations among the three
mulberry species tested for rutin manufacturing. Adding auxins such as indole-3-acetic acid,
2, 4-dichlorophenoxyactic acid and naphthalene-1-acetic acid not only improved the growth
of callus and adventitious roots, but also increased protein and rutin content. Callus in the
culture of suspension generated more rutin in the presence of IAA than that in the lack of
IAA. However, in the lack of IAA, rutin secretion in a medium was higher. As a

22
consequence, when adventitious roots were cultivated in a normal full-strength MS medium
containing 5 mg/l IAA, the largest amount of rutin was generated.

Litz et al. (1995) created somatic proembryos from the callus about 3 months after
explanting onto crop development medium consisting of altered B5 formulations with 60g /l
sucrose, glutamine, arginine, asparagine, 2,4-dichlorophenoxyacetic acid with either 1.2 μM
or 4.6μM kinetin and 1.75g/l gellan gum. Germination happened in vitro where the
coleorhiza elongated and a tap root arose; however, retrieval of the plantlet was not proved as
the shooting axis did not elongate.

Chalupa et al. (1974) described the method of producing trees from the
undifferentiated tissue of poplar callus. On the modified Wolter and Skoog medium, the
finest root formation was noted when NAA was used as auxin in concentration 0.2 to 0.4
mg/l and cytokinins were omitted. In the lack of auxin and with 0.15 to 0.70 mg/l of BAP,
induction of leafy leaves from the undifferentiated callus was the most efficient on the
modified Linsmaier and Skoog medium. The best root growth at the basal end of excised
shoots was accomplished by transferring shoots to the sterile blend of perlit and sand with a
modified Wolter and Skoog medium.

Webb et al. (1989) grown zygotic Piceaglauca embryos were grown in the presence
of 2,4-dichlorophenoxyacetic acid, N6-benzyladenine, and sucrose varying from 0.5 to 4
percent for the induction of embryogenic callus and the development of stable embryogenic
callus lines. Embryogenic callus with all seedlots was caused from all three collections.
Embryogenic callus was caused at all concentrations of saccharose, but obviously 4% of
saccharose was lower, whereas general 1% was the best. Factors that favored embryogenic
callus induction also favored stable embryogenic callus lines manufacturing.

Chaudhury et al. (2000) explored tissue culture reactions of young Bermudagrass

cultivar' Tifgreen' and prevalent Bermudagrass cultivar' Savannah' were explored. When
cultivated with 4.52 to 13.57 μM (1–3 mg/l ) 2,4-D on the Murashige and Skoog medium,
youthful inflorescence sections produced unorganized non-embryogenic calli that had
lengthy tubular cells loosely connected on the surface. However, the incorporation of 6-
benzyladenine (BA) in the callus induction medium at 0.044 μM (0.01 mg l-1) caused the
development of a compact, nodular embryogenic structure on about 20% of the calli. The

23
embryogenic structure produced green plantlets with regeneration levels of 79.5 percent and
83.3 percent respectively for the two cultivars when young inflorescences lower than 0.75 cm
were grown. The green and morphologically normal were all 96 plants regenerated from calli
induced in the BA-containing medium.

Yasuda et al. (1985) grown coffea arabica leaf explants grown on average with 5
μm6-benzyladenine (BA) as the single plant growth regulator generated white friable calluses
forming somatic embryos. For more than 2 years, these calluses have been subcultured on the
same medium and are capable of producing somatic embryos.

Głowacka et al. (2010) studied were carried out on three M sinensis genotypes and
one M giganteus genotypes. Explants from the youngest inflorescences i.e. length 0.1–2.5 cm
showed a considerably greater level of callus induction than those from more advanced
inflorescences i.e. length 2.6–5 cm. Three out of the four genotypes tested showed the
greatest calli-initiated plant regeneration from the youngest inflorescences when grown with
5 mg/l 2, 4 dichlorophenoxyacetic acid (2, 4-D) and 0,1 mg l−16-benzyladenine (BA) on the
Murashige and Skoog basal media (MS). The percentages of calli from those regeneration
genotypes ranged from 45 to 76.7 percent, and the respective levels of shoot regeneration
ranged from 1.85 to 6.33 shoots.

Rueb et al. (1994) launched callus from mature Japonica cultivar rice embryos Taipei
309 depending on the medium used, the number, mass and morphology of the callus formed
on the scutellum. The frequency of embryogenesis and plant regeneration was increased by a
restricted humidity and ideal aeration of the culture vessels. As a consequence, 98% of the
T309 embryos formed callus, 63% of which regenerated into crops. The regenerants were
discovered to be normal in plant morphology, fertility and seed set.

Tao et al. (2002) induced the callus from Citrus grandis and Osbeck leaf explants on
MS medium accompanied by 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid,
2,4,5-trichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic, 4-methoxy-3,6-
dichlorobenzoic acid or 4-amino-3,5,6-trichloropicolinic acid. 2, 4-D was the most powerful.
Only green, compact 2, 4-D calli at small levels i.e. 0.9 and 4.5 μM were capable of shooting
and regenerated more than 13 shoots per callus on MS medium containing at approximately
6.66 μM of benzyladenine. A 5–7 shoot multiplication rate was accomplished with 0.89 μM

24
BA from the shoot tip culture on the MS medium. Roots developed with 9.84 μM indole-3-
butyric acid and 5.37 μM NAA when regenerated shoots were grown on MS medium..

Rani et al. (1999) launched callus cultures on the Murashige and Skoog medium from
axillary leaves, axillary shoots, hypocotyls and root segments, supplemented by 2, 4-D i.e. 2
mg l/l and KN i.e. 0.2 mg/l. Shoots best distinguished on MS medium containing BA i.e. 2
mg/l from axillary shoot base callus. Regenerated roots with IBA alone and with the
combination of IBA with IAA are best rooted on MS medium. Plantlets were transferred to
pots containing mixture of sand and soil, acclimatized in a cultivation room and then
transferred to the glasshouse.

Freytag 1988 conducted regeneration tests on six sugarbeet (Beta vulgaris L.) lines. A
medium containing MS inorganic salts supplemented with 0.4 mg/1 N6-benzyladenine, 0.1
mg/1 indole-3-butyric acid, ten vitamins and six amino acids, was superior for both
adventitious shooting and callus formation. It took 4 to 6 weeks from explants to rooted crops
to finish the process of regeneration through adventitious shooting. When moved to the new
medium, the callus that formed on non-organogenic petioles was regenerative. Callus
regeneration happened primarily through low frequency shoot organogenesis but also
through somatic embryogenesis.

D'Souza et al. (2001) regenerated plantlets with 4.9 μM 2-isopentenyl adenine from
shooting apex and nodal explants on the B5 medium. The shoot apex explant growth factor
was greater about nine shoots per explant than that of nodal explants which were five shoots
per explant. Regenerated roots from shoot apex explants were best rooted on MS medium
supplemented with 0.05 μM α-naphthalene acetic acid. Whereas nodal explant regenerated
shoots required 2.7 μM of NAA for rooting. On the finest potting substratum, coco peat, 80%
survival of in vitro transferred crops happened. This protocol can be used for commercial
microprogation since the multiplication factor was nine per explant.

Shavalli et al. (2002) created a protocol for Bixa orellana L seed callus plantlet
regeneration. Seeds showed a large proportion of callus induction and a low yield of white
friable callus on the Murashige and Skoog (MS) medium comprising 5μM 1-
naphthaleneacetic acid and 2.5μM N6-benzyladenine within 6 weeks of dark cultivation. The
frequency of callus induction was higher than 16 h light/8 h dark photoperiod or 24 h light

25
photoperiod below 24 h dark. Shoots extended to 4 cm within 4 week of transferring to MS
without growth regulators. Shoots were rooted with a 5.0 μM (IBA) half-strength MS
medium. Approximately 85% of these crops were created after 3 weeks of hardening in pots
comprising pure garden soil and organic manure.

Garg et al. (1996) created a nodular callus on the MS medium from the immature
endosperm of Acacia nilotica by the addition to 2, 4-D, BAP and casein hydolysate,. The
callus distinguished somatic embryos in the third passage on this medium, in the dark. Only
after 15 d of pretreatment on altered MS medium the embryos germinated on MS in which
significant salts were substituted by those of significant B5 medium salts and came with
glutamine, casein hydrolysate and coconut water .

Thomas et al. (1999) generated unpollinated ovarian culture which has gynogenic
haploids of a female mulberry clone. Segments of inflorescences developed in vitro were
grown on MS plus BAP and 2.4-D and moved to MS with the combination of 2.4-D glycine
plus proline after three weeks. Seventeen percent of the ovaries created a gynogenic seedling
in this therapy. The gynogenic crops were founded in the soil. Of the 20 cytologically
examined gynogenic crops, twelve showed the haploid amount of chromosomes and the
remaining eight were aneuploids, with 13–17 chromosomes in their root tip cells.

Ortiz et al. (2000) used a procedure consisted of putting immature, zygotic embryos
in Murashige and Skoog semi-solid basal medium supplemented by 9.05 μM 2, 4-
dichlorophenoxyacetic acid and 4.65 μM kinetin to induce callus. Somatic embryos were
generated on semi-solid differentiating media without growth regulators or abscisic acid.
Somatic embryos germinated on average comprising adenine phosphate with an efficiency of
69% in A. Farnesiana and 47% in A. schaffneri. The greenhouse acclimatized some somatic
embryos that evolved into plantlets, and they grew into ordinary crops.

Xie et al. (2001) caused embryogenic callus on MS medium comprising TDZ, IAA
combinations and amino acid combination. Globular embryos created on the induction
medium cultivated on embryogenic callus. By a two-step maturation stage, almost 42 percent
of embryo cultures with globular embryos generated torpedo-and cotyledonary-stage
embryos. The first phase happened on basal half strength MS comprising 30 g/l sucrose and 5
mg/l GA3 followed by the second phase on basal 1/2-strength MS containing 50g / l sucrose.

26
Of the somatic embryos in the cotyledonary stage, 11% germinated into seedlings which
could be effectively transported to pots. This is the first effective crop regeneration report for
this economically significant tropical forest species through somatic embryogenesis.

Susheelamma et al. (1996) researched twenty-five mulberry genotypes were for callus
induction, to assess hormone efficacy in encouraging callus development, and to recognize
regenerating plant genotypes. Fifteen genotypes have shown initiation of the callus. For
longevity and the rate of development of callus cultures, genotypical variation was also
observed. For more than a year, calli of distinct genotypes have been retained. Callus
initiation frequency was high on the modified medium of Murashige & Skoog integrated
with 2, 4-D, casein hydrolysate and coconut water. Six genotypes suggesting genotypical
specificity have accomplished regeneration through organogenesis.

Rao et al. (1991) used acacia auriculiformis hypocotyl explants which were of 5 -10
mm in length and were excised in vitro from 10-day seedlings and grown on altered MS
medium with or in conjunction with KN, BAP, 2,4-D and NAA. The greatest morphogenetic
reaction was demonstrated by BAP and NAA with the combination of BAP. Glutamine
increased the proportion of calli displaying peaks and amount of shots per explant relative to
the lack of arginine and media. The callus organogenetic capacity decreased as the
concentration of sucrose in the MS medium rose from 30-50g /L; no shoots formed at 60g /L
and the callus turned black.

Das et al. (1997) obtained somatic embryogenesis of semi-solid Murashige and

Skoog (MS) salts and vitamins supplemented with 0.46–1.16 μM kinetin, 6.78–9.04 μM 2,4-
dichlorophenoxy acetic acid and 30 g/1 sucrose was obtained in callus cultures derived from
40-day semi-solid zygotic embryos of Dalbergia sissoo. Somatic embryos proliferated
quickly through secondary somatic embryogenesis following transition to a half-strength
basal MS medium supplemented with 0.46-1.16 μM kinetin and 6.78–9.04 μM 2,4-D with
2% sucrose. The light-green somatic embryos germinated with 0.5 mg/1 abscisic acid and
2% sucrose on half-strength MS salts and vitamins. Light and scanning electron microscopy
have researched the developmental phases of somatic embryogenesis.

Badji et al. (1993) have taken explants from crops that were either manufactured in a
sterile setting or in a greenhouse for four years. Zeatin or 6-benzylaminopurine (BAP) were

27
mixed at different concentrations with Murashige and Skoog's medium (MS) of which the
quantity of macroelements was divided in half. A two-stage process was required to achieve
a rooting rate of the small cuttings close to 100%. The first stage, called induction, consisted
of leaving the cuttings for 6 to 12 d on a Jordan medium which reduced the amount of
macroelements by half and added NAA at a concentration of 5.010−5 M. The second phase,
called root extension, needed this second hormone-free medium to plant the tiny cuttings.
After a few days, roots emerged. In a greenhouse, acclimatization occurred at a survival level
of nearly 100% when the rooted in vitro crops were transplanted into pots comprising a
combination of vermiculite and top soil.

Nandwani et al. (1995) noted shoot bud formation from cotyledonary nodes on the
medium of Murashige and Skoog (MS) comprising different doses of cytokinin viz 6-benzyl
adenine and kinetin, with or without auxin viz 1-naphthalene acetic acid and indole acetic
acid. In addition to NAA i.e. 0.1mg/l and BA i.e. 5.0 mg / l, multiple shoot regeneration
about 13-15 was accomplished on MS medium. Incorporation of auxin, for example,
promoted callusing in the explants by IAA and NAA. With any therapy used, no regeneration
of shoot buds from hypocotyl explants has been noted. Half-strength MS medium containing
indole butyric acid (3.0 mg/l) in regenerated shoots was discovered appropriate for rooting.
Plantlets created in vitro have been transmitted to soil.

Ahmad et al. (1991) segmented nodal explants from separate stem positions of
germinated seedlings to about 2 to 4 mm long and grown at separate concentration
concentrations on Murashige and Skoog basal medium supplemented with 6-benzylamine
purine (BAP) and kinetin. The MS basal medium with 0.5 mg/l BAP was the best mixture to
induce a greater multiplication of shots with an average of 25.4 shoots per explant. For root
formation, 85.0 percent rooting proportion of excised shoots treated with Seradix 3 which is
commercial rooting powder.

Kaur et al. (1998) accomplished maximum shoot bud growth i.e. eight to ten from a
single explant by the combination of 6-benzylaminopurine i.e. 4.0mg/l and α-
naphthaleneacetic acid i.e. 0.5 mg/l supplemented on Murashige and Skoog medium.
Addition to the medium of adenine sulphate i.e. 25mg /l, ascorbic acid 20mg/l and glutamine
150mg/l was found to be beneficial for maximum induction of the shoot bud. The shoot buds

28
evolved into healthy, sturdy shoots with BAP and kinetin at 1.0 mg / l on the MS medium. In
order to achieve full crops, excised shoots were rooted in half strength MS medium with 3mg
/l indole-3-acetic acid and 1.5 percent sucrose.

Galiana et al. (1999) juvenile explants were grown on Murashige and Skoog (MS)
medium comprising 1mg/l or 2mg/l benzyladenine i.e. 2-week-old of 5-to 10-mm length
consisting of two leaves. BAP levels used in the multiplication medium had a major impact
on the rooting capacity of juvenile explants. When both kinds of explants were multiplied on
an MS medium containing 1mg/l BAP and moved to a half-strength MS medium containing
0.05mg/l IBA, only 10% of juvenile explants were established compared to 70% of the 7-
month-old explants. When inoculated with a particular strain of Bradyrhizobium sp., rooted
crops transmitted to artificial substrates were all nodulated.

Bhaskar et al. (1998) used nodal explants of an 8-yr-old elite tree, and numerous
shoot proliferations were accomplished in A mangium. The highest amount of shoots was
achieved on BA 3mg/l and NAA 0.1mg/l supplemented MS medium. Adding ascorbic acid
to the medium of shoot proliferation inhibited medium growth. In vitro regenerated shoots
were rooted in IBA and IAA supplemented MS medium. After hardening, genotypically
uniform regenerated plantlets survived (70%) in the soil.

Jain et al. (1992) obtained callus from pre-soaked internodal explants of mulberry
comprising 2, 4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid and BAP on Linsmaier
and Skoog medium. Explants soaked in low BA concentrations for 48 to 72 h generated loose
and nodular callus that demonstrated the capacity to regenerate. Calluses created adventitious
shoot buds on average BAP within 3–4 weeks. Fifteen-week-old calluses had less shooting
buds than five-week-old calluses. Development of ordinary shoot bud primordia was high in
youthful calluses owing to sub-surface reorganization. The decline in the frequency of
primordia shoot bud formation with callus ageing is due to decreased activity of cell division
in both epidermal and sub-epidermal layers.

Jain et al. (1996) examined the anthers of Morus indica L. which were cultivated with
microspores at the uninucleate level; and the effect of temperature and kinetin pretreatment
on the formation of androgenic callus. The anthers divided and formed embryogenic calluses
on the MB medium supplemented with NAA and BAP using 8% sucrose. Rhizogenesis was

29
caused with decreased myo-inositol supplemented with NAA and BAP. When calluses were
transferred to the medium accompanied with NAA, BAP , 2,4- D and PVP different types of
embryos were launched. These embryoids further created roots on removal of 2, 4-d from the
medium and evolved without cotyledons precociously and created elongated shoots.

Thomas et al. (2000) grown immature endosperm of Morus alba on Murashige and
Skoog medium comprising 5 μm 2,4-dichlorophenoxyacetic acid, a continually increasing
callus was acquired. Shoot buds were produced on a medium containing cytokinin or
cytokinin and1-naphthaleneacetic acid (NAA) when the callus was subcultured. On the
medium comprising thidiazuron (1 μM) or benzylaminopurine (5 μM) and NAA (1 μM) the
highest amount of shoots was created. Shoots were multiplied and rooted in vitro by forced
axillary branching. Plants derived from Endosperm have been created in soil

Thomas et al. (2009) standardized an effective micropropagation protocol based on

various shoot induction and callus regeneration in Sarcostemma brevistigma, a rare medicinal
plant. The nodal cuttings were grown alone or in conjunction with NAA on MS medium
supplemented with BAP i.e. 0.5–8 μM or Kn 0.5–8 μM. On MS medium supplemented with
4 μM BAP, maximum various shoot induction was noted. The callus was subcultured alone
or in conjunction with NAA for shoot organogenesis on MS medium supplemented with BA
or Kn. The plants acquired through various shoot induction and organogenesis was rooted on
a half-strength MS medium supplemented with NAA or IBA. IBA was better than NAA,
both in terms of the proportion of responding crops and the average amount of roots per
explant. With 86 percent achievement, the rooted shoots were effectively transplanted to soil.

Oka et al. (1981) obtained Murashige and Skoog's medium cultivated leaf explants of
mulberry from aseptically cultivated shoots and seedlings. By varying the concentration of
benzyladenine, normal and abnormal leaves were cultivated. They differed in the formation
of adventitious buds. In normal leaves bud initiation occurred after removing petioles
exclusively at the cut ends of midribs, while in abnormal ones buds formed on midribs and
petioles in any region.

Chitra et al. (1999) noted a high frequency of sprouting about 80 % in the main nodal
explant cultures of Morus indica L, and shoot differentiation. Cultivar M-5 supplemented by
2,4-D on MS medium. In vitro proliferated shoots were quickly multiplied with BAP culture

30
of shoot advice on MS medium, producing the largest multiple shoot formation.
Multiplication was also achieved by cultivating shoot tips on MS medium with BAP and
GA3 which facilitated shoot elongation followed by sprouting in vitro grown shoots of
axillary buds.

Hanzel et al. (1985) assessed ninety-one genotypes of barley for the initiation of
growth of callus and its maintenance. Callus cultures were preserved at monthly intervals by
subculturing on new media. Murashige and Skoog's (MS) media promoted more callus
initiation and improved callus maintenance than B5 and Shenk and Hildebrandt media.
Callus initiation frequencies were high when adding either 0.5, 1 or 2mg/l of 2,4-D to the
media. Established calli were transmitted to differentiation media to support plant
regeneration.

Abe et al. (1986) used sixty varieties of rice (Oryza sativa L.), belonging to three
subspecies, japonica, indica and javanica including some hybrids were likened for their
ability to grow callus and regenerate plants. Tissue cultures launched from mature plants on
the 2mg /l 2,4-dichlorophenoxyacetic acid medium of Murashige and Skoog have been
transmitted to a medium comprising kinetin from which plantlets have been regenerated.
Most japonica varieties created a callus weighing more than 100 mg per seed 30 days after
inoculation and showing comparatively elevated regenerative capacity, whereas indica
varieties, japonica-indica hybrids and javanica varieties showed bad callus development and
plant regeneration.

Takamura et al. (2004) produced embryogenic calli from longitudinally bisected

sections of Cymbidium Twilight Moon a hybrid orchid, supplemented with NAA or 2,4-D
alone or in conjunction with TDZ within one month. For callus formation, the medium with a
mixture of 0.1 mg/l NAA and 0.01mg/l TDZ was ideal. Calli proliferated very well when
subcultured on the same medium every 4 weeks. Callus-derived PLBs transformed after
approximately 4 months into ordinary crops with well-developed shoots and roots on the
medium without plant growth regulators, which were acclimatized in the greenhouse at 100%
survival pace.

Parimalan et al. (2008) inoculated different kinds of explants: nodal shoot tips, shoot
tips and single nodes from in vitro cultivated seedlings on the Murashige and Skoog (MS)

31
medium supplemented by separate plant growth regulator levels and combinations. Up to
31.1μM N6-benzyladenine and 14.7 μM phenylacetic acid were inoculated on MS medium
with the highest amount of stem buds. Subculturing of the shoots on the MS medium
accompanied by BA and PAA resulted in enlarged shoots. Optimum rooting was achieved
when elongated shoots were inoculated on the MS medium supplemented with 4.9 μM
indole-3-butyric acid (IBA). The rooted plantlets were hardened and after 6 weeks their
survival rate was 73%.

Primalan et al. (2009) accomplished an effective annatto micropropagation protocol

by using nodal shoot tip explants. In addition to multiple levels and combinations of indole-
3-acetic acid N6-benzyladenine and triacontanol, shoot buds were acquired on the Murashige
and Skoog (MS) medium. MS media containing 0.05 μM IAA, 8.87 μM BA, and 11.2 μM
TRIA generated a maximum of 213 shoot buds along with 18 main shoots. MS medium
comprising 6.66 μM BA and 2.45 μM indole-3-butyric acid was best elongated by main
shoots. In addition to 4.9 μM IBA, the regenerated shoots rooted best on MS medium.

Gamborg et al. (1997) grown embryos with 5 μM 2,4-dichlorophenoxyacetic acid

(2,4-D) and 10–50 μM zeatin on a specified agar medium containing Murashige-Skoog (MS)
mineral salts. The callus was manufactured on the same medium within 10 weeks and
proliferated further after subculture. Small parts of the callus with leafy shoots were
transmitted to the same nutrient medium but containing indoleactic acid (IAA) of 1–1 μm.
Manufactured plantlets were transferred to pots after 8–10 weeks and grown to maturity.
Some crops generated fertile seeds while others were sterile.

Hammerschlag et al. (1985) acquired a white, nodular, extremely regenerative callus,

when friable main callus were transmitted from immature embryos to media comprising 4.5
μM 2,4-dichlorophenoxyactic acid and 0.44 μM benzyladenine (BA) comprising 0.27 μM α-
naphthaleneactic acid (NAA) and 2.2 μM BA. Maximum shoot regeneration occurred when
highly regenerative callus were transferred to a medium where the concentration of NAA
was reduced five times and the concentration of BA was increased twice. Regenerated shoots
were rooted on a 28.5 μM indoleacetic acid medium in the dark.

Ozgen et al. (1996) Immature embryos were dissected aseptically from seeds and
placed in plates containing Murashige and Skoog's (MS) mineral salts and 2 mg 2,4-

32
diclorophenoxyacetic acid (2,4‐D) per liter with the scutellum upwards. On 2,4-D-free
medium, calli and regenerated crops were preserved. Regenerated plants from both embryo
cultures were vernalized and cultivated to soil maturity. Callus induction rate and callus
regeneration ability were autonomous. Mature embryos have a low callus induction
frequency but a large ability for regeneration.

Bartok et al. (1990) achieved regeneration of feasible crops was in soybean using
callous derived from immature embryos. Regeneration happened via embryogenesis or
organogenesis depending on the structure of the medium. Embryogenesis occurred when
embryos were plated on the 43 μM α-naphthaleneacetic acid medium of Murashige and
Skoog (MS). Adding 30μM of nicotinic acid to the MS medium increased embryogenesis to
76%. For at least 12 months and 12–15 subcultures, the crops maintained the capacity to
regenerate full crops. Seeds were acquired from several regenerated crops and these
generated fertile crops that appeared normally when cultivated in the field.

Sharma et al. (2000) used regenerated shoots from axillary bud explants, a protocol
for micropropagation of mature Eucalyptus tereticornis Smith was created. Based on their
improved growth speed, physical and phenotypic features, and disease-free selection of trees.
In altered Murashige and Skoog medium, regeneration was acquired. During March–April,
regeneration from main explants was maximum. Subcultures took place every four weeks.
Hormone and media composition effects have been researched on regeneration and
development. Half-strength, altered MS medium and 1mg/l indolebutyric acid were the best
rooting.

Cannas et al. (1988) regenerated olive tree plantlets from the calli section of
cotyledon on a modified olive medium supplemented by 2iP alone or in conjunction with
indol-3-butyric acid. Calli were then transmitted to the same medium with distinct IBA
and/or 2iP concentrations to encourage further growth and acquire either root or shooting
calli bearing. On the OMc medium, the peak rooting was caused by 1 mg/l of IBA, while the
shoot induction was increased when the medium was supplemented by 4 mg/l of 2iP. Whole
plantlets were acquired by stimulating the regenerated leaves with 1 mg/l of IBA or
naphthaleneacetic acid to generate adventitious roots.

33
 Rai et al. (1988) created a good method for the multiplication of Indian rosewood in
vitro mass. In vitro plantlet regeneration was accomplished on the medium of Murashige &
Skoog comprising NAA and BAP from shoot tips and shoot sections of more than 50-year-
old elite trees. Regenerated calli stems were excised for rooting and handled with the
medium of Murashige & Skoog for the first time, supplemented by indole-3-acettic acid,
indole-3-butyric acid and NAA for 48 h to 72 h for the first time. After this therapy, plantlets
were transmitted to a hormone-free medium of half-strength MS. In the greenhouse, rooted
plantlets were transmitted to pots and cultivated.

Rao et al. (1987) created callus cultures have been from hypocotyl, leaf and stem
tissues of the tree legume Albizia lebbek Benth on modified Murashige and Skoog medium.
On callus medium with NAA and benzyladenine or kinetin combinations was a high
frequency of various buds and shoots. The rooting of the roots was induced with indole-3-
butyric acid (IBA) in Bonner's salt solution. From each explant, thirty to forty plantlets were
regenerated in an eight-week period. In the soil, establishment of plants have done.

Barve et al. (1993) micropropagated the chosen tree Commiphora wightii with BA
and kinetin by means of compulsory axillary branching on MS medium. In addition to BA,
kinetin, glutamine, thiamine HCL and activated charcoal, the highest frequency of shoot
formation was accomplished on MS medium. Transfer of shoots to a medium containing
reduced BA and kinetin concentration before rooting significantly stimulated shoot
elongation. Shoots could be rooted in the dark by treating both indoleacetic acid and
indolebutryic acid for 24 h and transferring them to a low-salt basal medium with activated
charcoal. The successful establishment of regenerated plantlets in soil.

Litz et al. (1985) reported about the production of tropical fruit which exceeds from
the temperate fruit production. Tropical fruits in many nations are not just significant
elements of the diet, but their export is a significant source of revenue. This applies
especially to those fruit plants that can be cultivated on a large-scale planting basis, e. G.
Bananas, papayas and so on. Exporting crude secondary products that can be recovered from
certain tropical fruits, e, generates additional export revenue. G. Papaya papain. Thus,
tropical fruit manufacturing not only improves the quality of life in tropical regions with few

34
recoverable natural resources, but also offers a precious and often irreplaceable source of
revenue.

Sita et al. (1986) reported the clonal spread of Indian rosewood (Dalbergia latifolia
Roxb.) from 5-year-old trees shoot callus cultures. In addition to BA and NAA, Bud
regeneration was acquired on MS media. Approximately 35% of the crops showed
organogenesis. Shoots measuring approximately 3–5 cm can be excised and supplemented
with 1–2 mg/l IAA in the medium of White. Rooted crops were created effectively in the
soil.

Arya et al. (2000) launched the embryogenic calli by using female gametophytes
comprising immature pre-cotyledonary embryos from Pinus roxburghii embryonic explants.
At multiple phases of development, Zygotic embryos were gathered and cultivated on
multiple media. Embryogenic calli initiation was accomplished in pre-cotyledonary zygotic
embryos with an embryonic head of 0.1-mm to 1.2-mm on B5 medium supplemented with
2,4-D or NAA and BA. Somatic embryos created with 5 μM 2,4-D or 10 μM NAA for stage-
I and stage-II embryos on the DCR medium. The early stage of callus growth was dominated
by a pro-embryo with six to eight meristematic cells and a suspensor of six to ten lengthy,
vacuolated cells.

Confalonieri et al. (2003) reported about the Populus species and hybrids which were
intensively grown in temperate regions of the globe as sources of woody biomass for forest
products sector and lowland reforestation. However, for classical breeding and selection, the
lengthy generation of trees, the presence of seasonal dormancy and the extended period
needed to evaluate mature features are powerful constraints. The combination of molecular
and classical breeding methods will assist to produce forest trees with beneficial
environmental impacts.

Shoemaker et al. (1986) assessed callus initiation and maintenance, seventeen cotton
cultivars were using 3 initiation media and 3 maintenance media. Calli were put on a 3
percent sucrose medium after a sequence of transfers of a 3 percent glucose medium. They
recognized cultivars Coker 201 and Coker 315 as embryogenic. Since then, in 6 weeks,
embryogenic callus has been regularly acquired by starting callus on glucose media for 3,4
weeks, followed by transfer to saccharose media. Transfer to auxin-free media with

35
decreased concentrations of sucrose at maturity outcomes in germination of the embryo.
Within 90 days of callus initiation, regenerated crops can be transmitted to the greenhouse.

Davidonis et al. (1983) defined the regeneration of cotton callus crops (Gossypium
hirsutum L. cv. Coker 310). Somatic proembryoids spontaneously created on a modified
Linsmaier and Skoog medium after two years of culture. Prolonged culture without
naphthalene acetic acid and kinetin increased the proportion of calli-forming proembryoids to
about 30%. Proembryoid development has been improved by the transfer of proembryoids to
non-NH4NO3 media but double the normal concentration of KNO3 and gibberellic acid
(GA). Promoted root initiation and development by reducing the concentration of glucose to
5g/1.

Gawel et al. (1986) a variety of media, leaf disks of four species and petioles of six
strains of Gossypium hirsutum were grown. Callus developed on all media from explants,
although it was extremely specific to embryogenesis. Embryos were created from only three
combinations of strainx media. A tiny proportion formed into plantlets of these embryos.
These findings show that cotton crops can be acquired from explants of leaf tissue.

Sears et al. (1982)launched tissue cultures on a modified Murashige and Skoog

medium containing 1mg 2,4-dichlorophenoxyacetic acid (2,4-D)/liter from immature
embryos about 12 days old. Cultures with 0.5 mg 2,4-D / liter were retained on the same
medium. Shoots were launched by decreasing the 2,4-D to 0,1 mg / liter and transferring full
crops to 2,4,-D-free media was regenerated. After four subcultures, eighteen genotypes were
able to regenerate crops.

Delporte et al. (2001) separated embryos were through a sterile nylon mesh from
surface-sterilized mature caryopses and grounded to bits. They were used as explants to
initiate embryogenic calli, supplemented with 10 μM 2,4-dichlorophenoxyacetic acid, on
strong medium. The morphogenic pathway of the initiated calli was followed for a 40-day
culture period. Out of 100 divided embryos, several hundred calli were generated within 3
days. A 90% callus induction rate was obtained and the 8th day of society emerged as
proembryos. The largest 47 percent embryogenic calli induction rate was achieved by
suppressing 2,4-dichlorophenoxyacetic acid after an induction period of 3–4 weeks. Finally,
two techniques of regeneration were compared.

36
 Munazir et al. (2010) explored effects of variable growth regulator levels including 2,
4-D, BAP, indole-3-acetic acid and kinetin were for main callus induction, embryogenic
callus formation and regeneration of two elite wheat varieties, Sahar and GA-02. Mature
seeds have been used as main callus induction explants. Maximum embryogenic callus
formation was noted with 0.1 mg / l IAA in conjunction with 1.5 mg / l BAP in the MS
medium, resulting in 73.51 percent and 62.33 percent embryogenic callus formation in the
Sahar and GA-02 respectively.

Mehmood et al. (2013) explored tissue culture reaction of 6 wheat cultivars for callus
induction and regeneration using MS and N6 medium supplemented with distinct
concentrations of 2, 4-D and BAP respectively. As explants, mature seeds were used.
Compared to the N6 medium, all cultivars had the best response for callus induction and
regeneration on the MS medium. Inqalab-91 and Lasani-08 showed peak callus induction
(90%) and (78.78%) of 2, 4-D at 3mg / l respectively. Tatara showed 84.43 percent callus at
2.0mg / l, chakwal-97 77.08 percent at 2.5mg / l, whereas GA-02 and Khyber showed 74.30
percent callus induction reaction and 65.97 percent callus reaction at 3.5mg / l 2, 4-D,
respectively. With regard to regeneration, the growth of immediate shoots and roots was
noted using various BAP concentrations. It was also noted that all cultivars showed shooting
with 3 and 5mg / l of BAP as well as root growth

Mendoza et al. (2002) conducted study to enhance the induction of callus and plant
regeneration from cultivar' Bobwhite ' wheat mature embryos. Effects of four auxins 2,4-
dichlorophenoxyactic acid , 3,6-dichloro-o-anisic acid ; 4-amino-3,5,6-trichloropicolinic acid
and 2-methyl-4-chlorophenoxy propionic acid (2-MCPP)] and effects of maltose vs. sucrose
were assessed under filter sterilized and autoclaved circumstances. All auxin ups with the
exception of 2 MCPP led in callus induction. Dicamba (18μM) in relation to 2.4-D resulted
in a double increase in the number of regenerated plants per embryo and reduced the time
required for plant regeneration by 3–4 wk.

Ozias et al. (1983) extracted tissue cultures from the scutellum of Triticum aestivum
L immature embryos resulted in somatic embryos with a well-defined scutellum and
coleoptile, as well as one or more primordial shootings and a root primordium. The ordinary
somatic embryos were created from compact white callus tissue that was not observed until 4

37
weeks or more after the initiation of culture. Murashige and Skoog's medium acquired the
largest frequency of white embryogenic tissue formation and the most ordinary embryoids
with twice the concentration of inorganic acids plus sucrose, inositol, casein hydrolysate,
glutamine and 2,4-dichlorophenoxyacetic acid.

Mokhtari et al. (2013) created a short-term high-efficiency protocol for direct shoot
regeneration and multi-shoot regeneration induction for in vitro plant genetic transformation
or propagation. Without callus formation, this tissue culture and regeneration scheme
requires about 2 months to complete, shorter than any of the processes available needing 3-4
months. Cutted embryonic meristem shooting , scutellum embryonic meristem shooting,
embryonic meristem shooting and scutellum, were removed from immature embryos and
cultivated in DSR medium. In some of the origins of explants, rapid inductions of direct
shoot buds and various shoot regeneration were obtained. On modified MS medium
containing 2 mg/l 2, 4-D and 10mg BAP, the highest reaction for various shoot regeneration
from explants was noted.

38
CHAPTER-3

MATERIALS AND METHODS

This research work was conducted at the Transformation lab, Center for Advanced
Studies,University of Agriculture, Faisalabad during 2017-2019.The objective of this study
was the “ Improving regeneration in shisham ( Dalbergia sisoo Roxb.)”

3.1 Materials:

3.1.1Chemicals and consumables

Stocks of certain chemicals were prepared for the future convenience like..

 2,4 D(2,4-Dichlorophenoxyacetic acid),

 Indole Acetic Acid (IAA),
 Benzylaminopurine BAP
 Naphthalene Acetic Acid ( NAA)
 Kinetin
Ultrapure water ( D3H2O) was used for making of all the stock solution
of different harmones which were used for the preparation of three different callus induction
media and three different regeneration media. Other required chemicals were:

 Murashige and Skoog (MS) with vitamins

 Murashige and Skoog (MS) without vitamins
 Sucrose
 Gellen gum, etc.

Several instruments such as :

 Weighing balance
 Hot plate and Magnetic Stirrer
were used for the preparation of media and stock solutions. Glasswares
which were used included:

 Measuring Cylinders

39
  Test Tubes
 Glass Beakers
 Flasks
 Culture bottles with lid
 Glass plates
The other things used for these experiments were:

 pH meter
 Dispenser
 Tweezers
 Scissors
 Forceps
 Scalpels
 Petri plates
 Refrigerator
 Oven
 Pipettes
 Vortex meter
 Falcons

3.2 Methadology:

3.2.1 Sterilization of media

Autoclave was used for the sterilization and disinfection of media and glassware at
1210C, and 15Psi, for 20 minutes to sterilize all consumables, Media and Solutions before
use. Horizontal Laminar Air Flow Cabinet was utilized for the sterilization of seeds, for
callus induction and for regeneration purpose.

3.2.2 Source of Plant Material

Seeds and green pods of Dalbergia sisoo will be collected from the Punjab
Forest,Research Institute and University of Agriculture,Faisalabad from the specific trees.

40
3.2.3 Surface Sterilization:

The ears of Horizontal Laminar Air Flow Cabinet were surface sterilized according to Frame
et al., 2002. Husk and bract was removed from the ear and washed with 70% ethanol and
soaked in the 10% fresh bleach solution for 3min (Almeida et al., 2004) then rinse with the
sterile distilled water for 2 to 3 times.

3.2.4 Sterilization of test tubes:

Test tubes were washed with the tap water. After washing bleach or spirit was
sprayed in test tubes and placed test tubes for 20mins. After 20mins these test tubes were
washed by cleaning test tubes with brush. After this, test tubes were placed for drying. After
drying, test tubes were autoclaved for use.

3.2.5 Media Sterilization:

Media prepared for germination was sterilized in autoclave at 1210c and 15Psi for 20
minutes.When media was in slightly warm situation , it was poured either in test tubes or
petri plates and covered with cling film. These media plates were noticed for few days just
for checking any type of contamination. During the pouring of callus induction media and
regeneration media, when media was slightly warm then required harmones were added, as
the efficiency of harmones decreases at high temperature.

3.2.6 Pre requisite for the culture of explants:

The Laminar air flow cabinet was sterilized by illuminating the ultra violet radiations
for 15 to 20 minutes before starting sterilization of seeds. Furthur sterilization was done by
using the 70% ethanol. Before culturing all the equipments were sterilized by autoclaving
and scalpels,forceps were surface sterilized by burning them to red hot conditions to avoid
any contaminations.

3.2.7 Sterilization of seeds:

Green pods and mature seeds were taken in a flask and washed at least three times
with the autoclaved distilled water to remove any debris or dust. After washing with distilled
water, seeds or green pods were washed two times with ethanol, and then further sterilization
of the plant material was done by adding the few drops of tween 20 along with 0.1%
mercuric chloride and shaking was done until scum was formed and then scum was removed
41
by washing it with autoclaved distilled water. Seeds or green pods were washed with distilled
autoclave water until all scum was washed out and then the plant material was taken out from
the flask and were placed on autoclaved filter papers which were arranged on the petri plates
in such a position that every seed was separated from each other so that they can dried
properly and no moisture was present in them .

Fig. 3.1 Sterilization of seeds

3.2.8 Sterilizing solutions;

 Ethanol : 70%

 Mercuric chloride: 0.1 mg/ 100ml

3.2.9 Germination of seeds:

When seeds and green pods become dry they were transferred to the autoclaved test
tubes or petri plates which contained half strength MS for the germination purpose. All the
above steps were performed in laminar airflow. This whole process was carried out using
sterilized autoclaved scalpel, forceps and test tubes. Test tubes were wrapped properly and
tightly with rubber band in such a way that no air should enter into test tubes and the plates
were sealed with the cling film and placed in light for proper germination. Only one seed is
placed in each test tube and at least 20 seeds were placed in each petri plate. Germination of
plant material can take upto 15 days. Following table shows the concentration and contents
of medium which was used:

42
Fig: 3.2 Separation of each seed to become dry

Fig: 3.3 Seeds were put in test tube for germination

Fig: 3.4 Germination of seeds in test tubes

43
 Table 3. 1 Recipe of the HALF MS Culture media

Contents Concentration

MS Salts 2.16g/L

Sucrose 30g/L

Gellen gum 2.68g/L

3.3 Optimization of callus induction protocol:

3.3.1 Callus induction:

Callus induction media was prepared by using full MS media.The pH of the media
was always adjusted to 5.7 to 5.8 before autoclaving. After autoclaving, media was cooled
down until became mild hot, at this point the required hormones specific to the callus
induction media which was used were added. Then each media was poured into test tubes
under aseptic conditions inside laminar air flow cabinet followed by wrapping them tightly
with rubber bands.

Seeds which germinate were dissected from their leaves and were cut into small
pieces with the goal that manipulation of callus was done quickly. Stems of the plantlets were
also cut for the induction of callus and placed on the callus induction media. After proper
labeling clearly mentioning media code, date on which work was done, the petri plates or test
tubes were transferred to growth room under the dark conditions at 280c. After 3 to 4 weeks,
formation of callus was observed from the small pieces of leaves, cotyledons, and stems.
Three different callus induction media were used for induction of callus which had only the
similar composition of full MS but have different hormones in them which were used to
check the best media for the induction of callus.

44
 Fig:3.5 Plantlets were dried before dessication

Fig:3.6 Cuttings of cotyledons to transfer on the callus media

45
Fig: 3.7 Callus induction on stem cutting

46
Fig: 3.8 Callus induction on callus induction media 1

47
Fig:3.9 Callus induction on callus induction media 2 and 3

48
Table 3. 2 Recipe for the Callus induction media 1:

Contents Concentrations

MS Salts 4.4g/L

Sucrose 30g/L

Gellen gum 2.68g/L

2,4,Dicholorophenoxyacetic acid 7.2mg/l

Kinetin 2.8mg/l

Table 3. 3 Recipe for the Callus induction media 2:

Contents Concentrations

MS Salts 4.4g/L

Sucrose 30g/L

Gellen gum 2.68g/L

Benzylaminopurine 19.8mg/l

Naphthalene Acetic Acid 5mg/l

49
 Table 3. 4 Recipe for the Callus induction media 3:

Contents Concentrations

MS Salts 4.4g/L

Sucrose 30g/L

Gellen gum 2.68g/L

Naphthalene Acetic Acid 5mg/l

2,4,Dicholorophenoxyacetic acid 7.2mg/l

3.4 Callus mass proliferation:

After 2, 3 and 4 weeks, callus mass proliferation was measured by using different
parameters like mass and weight. Then mass proliferation were categorized into low, very
low proliferation and good and very good proliferation.

Table 3. 5 Scale to score callus mass proliferation:

Callus Measuring Table

+
Very low proliferation
++
Low proliferation
+++
Good proliferation
++++
Very good proliferation

50
3.5 Plant Regeneration:

The equipment and material required:

 Callus
 Scalpels
 Forceps
 Blades
 Petri plates
 Regeneration Media
 Laminar flow
 Steri with glass beads
After two to three weeks of callus induction, calli were transferred to regeneration media
combinations. Three different regeneration media were used with combination of full MS
supplemented with Naphthalene acetic acid (NAA), Bromoaminopurine (BAP) ,Kinetin,
Indole Acetic Acid (IAA). Callus were cultured on this media were placed for two to three
weeks under light conditions.

Table 3. 6 Regeneration Media 1 (RM1) with MS salt:

Ingredients Concentrations

MS salts 4.4g/L

Sucrose 30g/L

Gellen gum 2.68g/L

Naphthalene Acetic Acid 2.68mg/l

Benzylaminopurine 19.8mg/l

51
 Table 3. 7 Regeneration Media 2 (RM2) with MS salt:

Concentrations
Ingredients

MS salts 4.4g/L

Sucrose 30g/L

Gellen gum 2.68g/L

Naphthalene Acetic Acid 2.68mg/l

Kinetin 21.3mg/l

Table 3. 8 Regeneration Media 3 (RM3) with MS salt:

Ingredients Concentrations

MS salts 4.4g/L

Sucrose 30g/L

Gellen gum 2.68g/L

Indole Acetic Acid 5mg/l

Benzylaminopurine 19.8mg/l

52
3.6 Statistical Analysis

The data was collected for callus induction media with at least 3 replications by the following
of completely randomized design. Analysis of variance and table was drawn and LSD was
used t o compare means and calculated for the treatment at 5% significance level.(Bari et al.,
2008).

53
Chapter 4

Results

This study was conducted at the Center for Advanced Studies in Food Security and
Agriculture, TAgriculture University, Faisalabad Transformation Laboratory. The research's
primary aim was to enhance the shisham regeneration protocol. Using tissue culture methods,
the skillful and reliable process of plant regeneration was expendable. The use of appropriate
explants, the use of appropriate media content and conditions of culture were of key
significance for the induction of embryogenic callus and regeneration faster than before and
achieved in the laboratory to enhance protocols for in vitro regeneration.

4.1. Preparation of Tissue culture

For this study project, a total of six distinct kinds of media were used. Three media
formulations have been intended to induce callus for the cultivation of green pods and mature
seeds from the Punjab Forest, Research Institute and Agriculture University, Faisalabad.
Callus induction media consisted of complete MS media with distinct harmone
concentrations particular to each media. For the regeneration protocol, another set of
complete MS media combinations supplemented with distinct concentrations of harmones
were used.

4.2. Callus induction

Green pods and mature seeds were grown on half MS media and moved to complete MS
callus-based media with combinations of callus induction media 1 (CM1), callus induction
media 2 (CM2), callus induction media 3 (CM3) after germination. Cotyledons were
originated and elongated after 3 to 5 days of growing green pods and mature seeds. After
being moved to callus induction media, removing cotyledons from the half MS then
encourages the initiation of callus formation. If the abaxial side of the leaf was placed down
then the more callus induction was seen while if the medium buried cotyledons then no callus
induction was seen.

54
The cotyledon was plated with an embryonic axis down position but in full contact with the
medium for elevated callus induction. The plates were put at 28 for about 15 to 20 days after
cultivation.

4.3. Callus induction

The callus induction rate was scored after a couple of days of callus initiation. The callus lev
el
was best at 2,4 D and kinetin, according to Rehman. To assess this hypothesis, for each medi
a, the rate of callus proliferation was scored and compared with each other in the following
table:

Table 4. 1 Analysis of variance table for No. of good proliferated callus

Source of Degrees of Sum of squares Mean squares F-value

Variation freedom

Media 3 5125.1 1708.32 164.99

Error 32 331.3 10.35

Total 35 5456.3

Callus proliferation data were gathered after 15 days of cultivation and it was observed that
the amount of very nice proliferated callus for callus induction media 1 was highly
significant other than two media callus induction media 2 and callus induction media 3.

55
Table of means:

Table of means showed that for callus induction media 1 the highest value was achieved
which supplied the very excellent proliferated callus among the three callus induction media.

Media Mean

Callus induction media 1 34.22±0.57

Callus induction media 2 26.78±0.72

Callus induction media 3 20.23±1.91

56
 40

No. of very good proliferated callus (15 days)

35

30

25

20

15

10

0
CM1 CM2 CM3
Callus induction media

Fig:4.3 (A) Graph of table of means of table 4.1

As shown in the fig 4.1 columns were denoting the rate of very good proliferated callus
while the vertical bars represent the means of the callus induction media, callus induction
media 1, callus induction media 2 and callus induction media 3.

57
 Table 4. 2 Analysis of variance table for No. of low proliferated callus

Source of Degrees of Sum of squares Mean squares F-value

variation freedom

Media 3 3408.2 1136.07 155.51

Error 32 233.8 7.31

Total 35 3642.0

Callus proliferation data was observed after 15 days of cultivation and it was observed that
the amount of low proliferation of callus was less recorded in callus induction media 1 and it
is high in callus induction media 2 and callus induction media 3.

Table of means

Media Mean±SE

CM1 12.56±0.70

CM2 21.63±0.37

CM3 27.71±1.58

58
Table of means has shown that maximum mean of callus of low proliferation of callus
induction media 3, callus induction media 2 and callus induction media 1 was 27.71, 21.63
and 12.56 respectively. It was observed that the callus induction media 1 provided the best
callus among callus induction media 1, callus induction media 2 and callus induction media
3.

35

30
No. of low proliferated callus (15 days)

25

20

15

10

0
CM1 CM2 CM3
Callus induction media

Fig: 4.3 (B) Graph of table of means of table 4.2

As shown in the fig the horizontal bars represent the type of media and the vertical lines
show the number of days of low proliferation callus and the data of low proliferated callus
was observed after 15 days.

59
 Table 4. 3 Analysis of variance table for No. of very good proliferated callus

Source of Degrees of Sum of squares Mean squares F-value

variation freedom

Media 3 4146.1 1382.03 285.53

Error 32 154.9 4.84

Total 35 4301.0

Data of callus proliferation was collected after 30 days of culturing and it was observed that
the number of very good proliferated callus was highly significant between the callus
induction media 1, callus induction media 2 and callus induction media 3.

Table of means

Callus induction media Mean±SE

CM1 31.67±0.65

CM2 21.89±1.183

CM3 15.01±0.47

60
Table of means has shown that maximum mean of callus of very good proliferation of callus
induction media 1, callus induction media 2 and callus induction media 3 was 31.67, 21.89
and 15.01 respectively. It was observed that the callus induction media 1 provided the very
good proliferated callus among callus induction media 1, callus induction media 2 and callus
induction media 3 and it was seen that low proliferation of callus was provided by the callus
induction media 3.

40

35
No. of very good proliferated callus (30 days)

30

25

20

15

10

0
CM1 CM2 CM3
Callus induction media

Fig: 4.3 (C) Graph of table of means of table 4.3

As shown in the fig 4.5 the horizontal bars represent the type of media and the vertical lines
show the number of days of very good proliferation callus and the data of very good
proliferated callus was observed after 15 days.

61
 Table 4. 4 Analysis of variance table for No. of very low proliferated callus

Source of Degrees of Sum of squares Mean squares F-value

Variation freedom

Media 3 2932.8 977.58 115.48

Error 32 270.9 8.46

Total 35 3203.6

Days of callus proliferation was collected after 30 days of culturing and observed that the
number of very low proliferation was highly significant among the three media callus
induction media 1, callus induction media 2, and callus induction media 3.

Table of means

Callus induction media Mean±SE

CM1 15.23±0.66

CM2 22.43±0.89

CM3 28.55±1.20

62
Table of means has shown that maximum mean of callus of very low proliferation of callus
induction media 3, callus induction media 2 and callus induction media 1 was 28.55, 22.43
and 15.23respectively. It was observed that the callus induction media 1 provided the best
callus among callus induction media 1, callus induction media 2 and callus induction media
3.

35

30
No. of very low proliferated callus (30 days )

25

20

15

10

0
1 2 3
Callus induction media

Fig:4.3 (D) Graph of table of means of 4.4

As shown in the fig 4.6 Columns were denoting the rate of very low proliferated callus while
the vertical bars represent the means of proliferation.

63
4.4 Type I and Type II induction of callus

On callus induction media 2 and callus induction media 3 whitish callus was observed that
was non embryogenic as shown in the figure 4.3:

64
 Fig.4.4 (A) Type I callus

After 2 weeks, on callus induction media the colour becomes to the yellowish and green that
was the embryogenic callus as shown in the fig.4.4

Fig.4.4 (B) Type II callus

65
4.5. Effect of 2,4-D , Kinetin , NAA and BAP
The rate of callus induction of mature plants relies primarily on media composition. The rate
of embryogenic callus relies entirely on the media concentration of 2-4 D. Using the 3
distinct callus induction media, the impact of 2, 4 D, kinetin, BAP, NAA on callus induction
from Shisham's immature embryos and cotyledons was assessed. There was an obvious
connection between the level of 2-4 D and the concentration of kinetin on the reaction of
callus induction. When grown on half MS mature seeds and green pods were
germinatee.After germination cotyledons were elongated aon half MS and were cut and
shifted to callus induction media 1, callus induction media 2, and callus induction media 3
and it initiated the callus formation. The rate of callus induction was differently seen as the
composition of media changed. The effect of all growth regulator harmones was shown in
graph. The callus induction media 1 contained 2,4 D and kinetin, giving the primary callus
and embryogenic callus more than others. The callus induction media 2 includes Benzyl
amino purine (BAP) and Naphthalene acetic acid (NAA) provides more primary but not
much embryogenic callus. The callus induction media 3 includes 2,4-D and NAA which
provides the lowest rate of primary callus and non-embryogenic callus.

66
 proliferation weight

4.5 1.8

4 1.6

3.5 1.4
Proliferation rate of callus

Average weight of callus

3 1.2

2.5 1

2 0.8

1.5 0.6

1 0.4

0.5 0.2

0 0
Light yellow Yellow Light greenish Greenish yellow
yellow
Health of callus

Fig: 4.5 (A) Callus proliferation on Callus Induction Media 1

All green pods and mature seeds were grown on callus induction media1 and originally
created the light yellow callus that after periods of weeks turns into yellow, light greenish
yellow and then greenish yellow this implies that the embryogenic calli was originally caused
by this media. With the passage of time, the rate of callus proliferation was high. The average
callus weight in the first week was 0.7 g, which in the next 4 weeks of cultured on this media
increased to 1.8 g. This rate of callus proliferation shows that 2,4-D and kinetin concentration
works as an optimum concentration for the induction and regeneration of plantlets into plants
for embryogenic callus.

67
 proliferation weight

3.5 1.4

3 1.2
Proliferation rate of callus
2.5 1

Average weight of callus

2 0.8

1.5 0.6

1 0.4

0.5 0.2

0 0
White Yellowish white Light yellow Greenish
yellow
Health of callus

Fig: 4.5 (B) Callus proliferation on Callus Induction Media 2

All green pods and mature seeds were grown on callus induction media 2 and originally
created the white callus that after periods of weeks turns into yellow white, light yellow and
then greenish yellow it means that this media initially induced the non embryogenic calli that
after two weeks become the embryogenic calli. With the passage of time, the rate of callus
proliferation was high. The average callus weight in the first week was 0.4 g, which in the
next 4 weeks of cultured on this media increased to 1.2 g. This rate of callus proliferation
shows that BAP and NAA play a role in the embryogenic callus induction and regeneration
but not more that callus induction media 1.

68
 proliferation weight

3 0.9

0.8
2.5
0.7
Proliferation rate of callus

Average weight of callus

2 0.6

0.5
.
1.5
0.4

1 0.3

0.2
0.5
0.1

0 0
Whitish yellow Yellow Light greenish Blackish yellow
yellow
Health of callus

Fig: 4.5 (C) Callus proliferation on Callus Induction Media 3

All green pods and mature seeds were grown on callus induction media 3 and originally
produced the light yellow callus that after intervals of weeks turns into yellow, light greenish
yellow and then blackish yellow it means this media initially induced the embryogenic calli
that after two to three weeks become dead. Initially the rate of callus proliferation was good
but after the two weeks the rate of growth of callus was stopped. The average callus weight
in the first week was 0.31g, which in the next 4 weeks of cultured on this media increased to
0.75 g and then not increased for next 4 weeks of culturing on this media not moved more.
This rate of callus proliferation showed that 2, 4-D and NAA were somehow good for the
induction of callus but didn’t good for the regeneration process. Callus induction media 1
was good for all perspectives related to colour, texture, weight, and regeneration capability of
callus.

69
4.6. Plant regeneration :
The plantlets capacity to regenerate into plants was associated with embryos capacity to form
callus. Callus was moved to the regeneration media after callus induction. Three
combinations with the corresponding concentration of harmones that were distinct from each
other were used for the shoot induction media. It was noted that reduced Naphthalene acetic
acid (NAA) concentration and greater Benzyl amino purine (BAP) concentration increased
the induction frequency of the leaves. Regeneration media 1 with full MS concentration
supplemented with BAP and NAA therefore showed best response because it contained high
BAP concentration and low NAA concentration. BAP-free media was not helpful in
encouraging differentiation. The plantlets have been regenerated from the embryogenic calli
after 4 to 5 weeks. Shoot induction was greater than RM3 (IAA and BA) on RM2 (NAA and
kinetin), but smaller than RM1. RM1 reacted by regenerating shoots per explant to the best
overall.

70
 Fig: 4.6 (A) Regeneration from callus

Fig: 4.6 (B) Subculturing of regenerated callus

71
72
Fig: 4.6 (C) Proliferation of regeneration phase in culture vessel

Fig 4.6 (D) Direct Embryogenesis of green pods on regeneration media

73
 Discussion

In this research, in colour, texture, amount, and organogenesis, callus induction from
cotyledons of green pods and mature seeds was distinct. There was a noticeable distinction in
the texture of the callus. Callus caused on cotyledons of green plants was soft, while the
induced callus on cotyledon of mature plants was mildly hard in texture. Compared with the
cotyledons of green pods, more organogenic callus was accomplished from the cotyledon of
mature seeds. The best response has been discovered for the NAA and BA medium. (Singh et
al., 2002) used semi-mature and mature cotyledon explants for adventitious shoot
organogenesis and plant regeneration of D.sisoo. Full MS with BA and NAA was best for
semi-mature cotyledon regeneration, whereas MS with BA without NAA was best for mature
cotyledon regeneration.

Chand and Singh (2005) regenerated plants of Dalbergia sissoo from the callus
cultures of semi-mature embryos. In conjunction with kinetin, callus was induced on MS
medium with 2,4-D, and shoot regeneration happened on MS medium containing BA and
NAA. Results in this research showed a better reaction with the mature seeds compared to
green pods. Using different 2,4-D and kinetin levels, Dalbergia sissoo accomplished somatic
embryogenesis and plantlet regeneration. Sujay et al. (2004) investigated five elite inbred
lines of maize using 14-day immature embryos as explants to induce and regenerate callus.
Explants cultivated on the medium of Murashige and Skoog (MS) supplemented with 2,4-
dichlorophenoxyacetic acid at demonstrated the greatest callusing frequency.

Bari et al. (2008) used Dalbergia sissoo nodal, internodal and shoot tip explants to
induce callus and regenerate the plant. Soft green callus was caused on nodal sections grown
on MS medium with BA and NAA, and the largest amount of shoots were regenerated on
MS medium containing BA and IAA. Thirunavoukkarasu et al. (2010) discovered that MS
medium was superior to woody plant medium for micropropagation of D. sissoo. In our
research, the best response for callus induction was obtained on MS medium with 2,4-D and
kinetin with mature embryos, and then cultivated for shoot regeneration on MS medium with
NAA and BA.

74
 Kumar et al. (1991) regenerated plants of D.sisoo from calli derived from cell
suspension of D. sissoo. Callus induction happened on MS medium cultured tissue with 2,4-
D and BA, and differentiation of the shoot buds occurred from cell suspensions plated on a
medium containing BA but without auxin. Pattnaik et al. (2000) acquired organogenesis
shooting and regeneration of plantlets from sissoo crops derived from hypocotyl cell
suspension. Huang et al. (2004) created an effective maize regeneration method by using
mature embryos and callus was triggered on the induction medium which was supplemented
by the 2,4-D.

Ali et al. (2012) made attempts for evaluating the best culture conditions for the
micro propagation of nodal meristem of Dalbergia sissoo. Within 10-12 days the media
which contain NAA +. BAP in MS media shows the best response of shoot formation and
after 25 days average, length of shoot was observed upto.2.4cm. Joshi et al. (2003) examined
the results of clonal propagation of Dalbergia sisoo on different nutrient media. Nodal
explants from the two cotyledons which were cultured on the two nutrient media MS and B5
and made with different combinations of the media. The media was supplemented with BAP
with the combination of NAA or IAA. NAA gives the maximum number of shoots and MS
media gives the best result for the proliferation of shoots in comparison with the B5 media.
Sharma et al. (1988) evaluated that when grown on Murashige and Skoog (MS) media
containing BAP + NAA, the hypocotyl explants of Dalbergia sissoo which were long
about one cm generated the shoot buds.

75
Chapter 6

Summary

Totipotency is the capacity of living cells to regenerate into a whole plant. This plant
cell capacity is used for the method of in vitro cultivation. An efficient technique for genetic
improvement of shisham against disease is the in vitro regeneration scheme. Biotechnology
is suggested as a remarkable alternative for adding ongoing work on genetically enhanced
germplasm development to achieve sustainable plant development.

The sterilized mature seeds and green pods of shisham were used as source of
immature embryos like cotyledons as explants and were cultured on half MS media for
germination. After germination cotyledons were cut and were placed on three callus
induction media of full MS which were supplemented with different concentration of
different harmones. Callus induction medium 1 was supplemented with 2,4-D and kinetin,
callus induction medium 2 was supplemented with BAP and NAA, and callus induction
medium 3 was supplemented with 2,4-D and NAA to get the callus .The response of different
harmones was different for the development of callus on different media. About 90%
embryos regenerated to produce the callus.

The callus induction medium 1 showed better response towards callus than other
media of callus induction. The three callus induction media showed response at different
intervals of time regarding the harmones. Results revealed that maximum score of average of
callus was found on the callus induction medium 1(CM1), while the minimum response was
found for the callus induction media 2(CM2) and callus induction media 3 (CM3) showed the
least response. Calli of 21 days or 30days were considered better for regeneration. After the
induction of embryogenic calli, calli were transferred to different regeneration media. The
best response was found for the regeneration media 1, which has lower concentration of
NAA and higher concentration of BAP.

76
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