CHAPTER 1

1.0 TITLE
An establishment of a reproducible system for Solanum tuberosum L. artificial seeds auxiliary
bud and shoots.

1.1 INTRODUCTION

Potato (Solanum tuberosum L.), the most important non cereal food crop in the world, is a
polyploid outbreeder which maintains a high degree of heterozygosity. Consequently, tetraploid
commercial cultivars are propagated vegetatively in order to maintain phenotypic characteristics
and economically important traits. Generally, propagation is via tubers from the previous crop,
but alternative clonal. propagation methods have been developed to accommodate emerging
economic production requirements. Tissue culture techniques, mainly micropropagation, have
substantially augmented the supply of planting material, and reduced the time required for the
release of new cultivars from more than 10 to 3–5 years (Struik and Wiersema 1999). Clonal in
vitro propagation methods not only maintain the uniformity among offspring, but also preserve
health status as the planting material has reduced exposure to soil-borne and other diseases.
Micropropagation techniques in potato are mainly employed to allow rapid mass propagation of
nuclear stock material, usually in the form of microtubers and axillary bud shoot plants.
Minitubers produced in initial cycles of multiplication from the in vitro material increase the
number of propagules in the worst year, and bulks up the prebasic seed required for large-scale
production of basic seed tuber material, which initiates the seed production programme. The
multiplication rate in microtuber and axillary bud based micropropagation systems depends upon
the number of pre-formed meristems present in explants during each multiplication cycle;
inherently, these numbers are low rendering such systems comparatively resource inefficient.
Somatic embryogenesis, however, the possibility of a novel system for the large-scale
multiplication of plant material and could be more economical because of the greater
multiplication rates, exploiting the potential of every cell to regenerate into a complete plant.

The encapsulation of somatic embryos to produce artificial seeds or synthetic seeds was first
proposed by Murashige (1977). According to Redenbaugh et al. (1987), synthetic seeds
consisting of somatic embryos were suggested as a powerful tool for propagation of plant
species. However, the production of synthetic seeds by encapsulating vegetative parts such as
shoot tips shoots primordial or axillary buds had been used in recent years as more suitable
alternative to somatic embryos (Sarkar and Naik, 1997). The seed consists of tissues derived
from vegetative parts or somatic embryos and artificial endosperms. The vegetative parts and
somatic embryos were coated by encapsulation matrix i.e., sodium alginate, subsequently formed
the artificial endosperm. Besides sodium alginate, polyethyleneimine (Kersulec et al., 1993) or
chitosane (Tay et al., 1993) were also used as encapsulation matrix. The condition of an artificial
endosperm could provide nutrients and growth regulators that were essential for plantlet
development from encapsulated parts (Nieves et al., 1998).

The synthetic seed is currently considered as the most effective and efficient alternate technique
for propagation of commercially important plant species that had problems in seed propagation
and plants produced non-viable seeds or without seeds. Its potential advantages include
stabilities during handling, potential for long terms storage without losing viability,
transportation and planting directly from (in vitro to field conditions and higher scale at a low
cost production (Ghosh and Sen, 1994). Few studies had been done on the production of
synthetic seeds from encapsulated vegetative parts of several plant species such as shoot tips of
banana (Ganapathi et al., 1992), nodal segments of Cassava (Danso and Ford-Floyd, 2003),
shoot buds of Morus indica (Bapat and Rao, 1987) and axillary buds of Daucus carota (Kitto
and Janick, 1985) with different degree of successes.

1.2 PROBLEM STATEMENT

Potato (Solanum tuberosum L.) is a temperate climate tuber crop and an important staple crop in
human diet in the world. The cost of production is very high since seed potatoes are very
expensive and accounts for more than 50% of the total cost of production. Pathogens such as
Ralstonia solanacearum, Streptomyces scabies, Erwinia spp. causes losses in potato crops
intensively and changes in the soil microflora has been listed as one of the possible causes of
productivity decline. The addition of herbicides can cause qualitative and quantitative alterations
in the soil microbial populations and their enzyme activities. Biological agents such as Bacillus
spp., Pseudomonas spp. and Trichoderma spp. can be used for treatment of sick soils. Main
limitation is due to the fact that the agents should colonise the roots in order to be active.
JUSTIFICATION

To overcome this bottleneck, this study attempts to produce synthetic seeds that in future studies
can be incorporated with an antagonistic rhizobacteria to produce an efficient bio-control
system.

Most communal farmers to access potato seed the rely on informal channels to access the seed
which is often low quality material. There are challenges with both the formal and informal
systems. Quality declared seed is 2% and certified seed 2-3%, so the bulk of the rest constitutes
the informal seed system. Controlling disease in the informal systems is very important. Seventy
percent of farms in Sub Saharian region with the exception of South Africa have bacterial wilt
due to farmer-to-farmer exchange of seed, underscoring the need to support the informal system
as well.

The production of seed potato is complex and is associated with several complications. Due to
increases in input costs such as fuel, electricity and labour for seed production, the return on
investment for some growers does not justify the risks associated with production. Biosecurity is
another important factor. Pests and diseases that can have a devastating impact on the industry as
potatoes are propagated vegetatively.

The use of synthetic seeds offers a wide range of advantages which range from short production
time to ease of handling. Other advantages include

Ease of handling while in storage

Easy to transport

Has potential for long term storage without losing viability

Maintains the clonal nature of the resulting plants

Serves as a channel for new plant lines produced through biotechnological advances to be
delivered directly to the green house or field

Allows economical mass propagation of elite plant varieties

1.3 AIMS AND OBJECTIVES

AIM

To establish an efficient and reproducible system for the production of synthetic seeds derived
from auxiliary bud and shoots

OBJECTIVES

To evaluate the effect of different concentrations of gelling agents in encapsulation

To evaluate the effect of preparing encapsulation matrix in different solutions

To evaluate the growth of the derived synthetic seeds of different sowing media

1.4 HYPOTHESIS

H 0: It is not possible to efficiently generate synthetic seeds derived from auxiliary buds and
shoots.

H 1: It is possible to efficiently generate synthetic seeds derived from auxiliary buds and shoots.
Chapter 2: Literature Review

2.1 ORIGINS AND HISTORY OF INTODUCTIONS

Solanum tuberosum ultimately traces its origin to Andean and Chilean landraces developed by
pre-Colombian cultivators. These landraces exhibit tremendous morphological and genetic
diversity and are distributed throughout the Andes, from western Venezuela to northern
Argentina, and in southern Chile. The wild species progenitors of these landraces have long been
in dispute, but all hypotheses centre on a group of approximately 20 morphologically similar
wild species referred to as the Solanum brevicaule complex (Correll 196; Grun 1990; Miller and
Spooner 1999; Ugent 1968; van den Berg et al. 1998).

The first record of S. tuberosum subsp. andigena outside South America was in the Canary
Islands in 1567 (Hawkes and Francisco-Ortega 1993; Ríos et al. 2007), and shortly thereafter in
continental Spain in 1573 (Hawkes 1990; Hawkes and Francisco-Ortega 1992; Romans 2005).
Forms of the introduced S. tuberosum subsp. andigena were adapted to the longer day lengths
and climate of European latitudes through selection (OECD 1997). These converted forms are
known today as S. tuberosum subsp. tuberosum (or S. tuberosum).

From Europe, S. tuberosum was transported to North America. S. tuberosum may first have been
transported from England to Bermuda in 1613 and then from Bermuda to the North American
mainland in 1621, a hypothesis favoured by Laufer (1938) and Hawkes (1990). S. tuberosum was
present in India by 1610 and mainland China by 1700 (Sauer 1993). S. tuberosum was taken to
New Zealand in 1769 by Captain Cook and gained agronomic significance for the native Maori
by 1840 (Sauer 1993). Missionaries may have played a crucial role in the distribution of S.
tuberosum from Europe throughout the world (Laufer 1938; Sauer 1993).

2.2REPRODUCTIVE BIOLOGY

Solanum tuberosum is a perennial but is grown as an annual in most parts of the world. The
commercial crop is propagated vegetatively using tuber pieces or small whole tubers that are
commonly referred to as seed or seed potatoes or through plant cuttings or plantlets. S.
tuberosum may also be reproduced by botanical seeds, which are commonly referred to as true
potato seeds or TPS. True potato seed production is practised in breeding programs under
greenhouse or growth chamber conditions. Some programs have also used open pollination
conducted outdoors. True potato seed production in the natural environment varies with cultivar
and weather conditions. The degree to which flowering occurs, the duration of flowering, and the
response of flowering behaviour to environmental conditions is greatly influenced by cultivar
(Burton 1989). The environmental conditions that influence flower initiation and development
include light intensity, quality and duration (day length), temperature, water supply, and
available soil nutrients. Flowers of some varieties may abscise prematurely.

Tetraploid S. tuberosum is self-compatible, although most of the related diploid species are self-
incompatible. Pollen sterility occurs frequently in S. tuberosum, and ovule sterility occasionally;
many varieties do not produce any botanical seed.

Flowering starts on branches located near to the base of the plant and proceeds upwards. Each
flower will typically remain open for 2 to 4 days, with the stigma being receptive and pollen
being produced for approximately 2 days (Plaisted 1980). Fertilization occurs approximately 36
hours after pollination (Clarke 1940). Viable seeds require a minimum of 6 weeks to develop.

Tubers are storage organs that develop from swollen underground stems, and the eyes on the
tubers are buds that can sprout and develop into new stems. New plants can be grown from
whole tubers or pieces of tubers, and the number of stems produced from a planted tuber or tuber
piece depends on the number of eyes and the physiological age of the tuber. The tuber acts as a
source of nourishment for the new plant, and plants grown from tubers tend to have more early
vigor than those grown from true potato seed (Hoopes and Plaisted 1987). Vegetative
propagation may perpetuate diseases in successive generations.

2.3 BREEDING AND SEED PRODUCTIONS

The majority of breeding with Solanum tuberosum involves crosses between tetraploid
genotypes followed by phenotypic recurrent selection (Carputo and Frusciante 2011; Dean
1994). Parents are selected to be diverse in order to minimize homozygosity and inbreeding
depression, and test crosses may be performed in order to determine which parent combinations
are desirable. Selection is typically applied at the phenotypic level, although molecular markers
are increasingly used (Bradshaw 2007; Carputo and Frusciante 2011). Due to the heterozygosity
and tetraploidy of S. tuberosum, traits are expected to segregate in the F1 generation, and large
populations are typically generated, on the order of tens of thousands (Carputo and Frusciante
2011; Howard 1978). From the F1 generation, tubers will be removed and planted, representing
the first clonal generation. The clones will then be put through a series of field trials in an
increasingly diverse range of environments over a number of years, and selection will be applied
to reduce the number of clonal lines until only one or a few remain (Carputo and Frusciante
2011; Dean 1994).

Breeding may also be done using diploid lines. Maternal haploid lines can be made by crossing
S. tuberosum with diploid clones of S. phureja (Carputo and Frusciante 2011). These can then be
used in crosses with 2x tuber-bearingSolanum species. Direct crosses between diploid tuber-
bearing Solanum species and tetraploid S. tuberosum may also succeed if the diploid can produce
unreduced (2n) gametes, which is common for these species (Hoopes and Plaisted 1987). Other
techniques such as mutagenesis, somatic hybridization, and genetic engineering may also be
employed (Bradshaw 2007; Hoopes and Plaisted 1987; Karp et al. 1987).

The main objectives of breeding include increased yield, improving quality characteristics of
tubers such as skin and flesh colour, tuber size and shape, eye depth, nutritional properties,
cooking/after cooking properties, processing quality, and introducing resistance to biotic and
abiotic environmental stresses (Carputo and Frusciante 2011; Howard 1978).

Seed potato production often occurs in regions that are separate from those used to produce the
crop for consumption. Precautions are taken during seed potato production to minimize disease
incidence (Dean 1994; Hoopes and Plaisted 1987; Western Potato Council 2003). Insecticides
and other insect control measures will be used to reduce aphid populations, which are the main
agents for spreading viral diseases. Any plants showing symptoms of disease are rogued;
rogueing may be supplemented with laboratory detection methods when symptoms may be
poorly expressed. Management and sanitation practices will also be put into place to minimize
the spread of disease through contact with machinery, tools or with surfaces encountered during
transport and storage.

In Canada, Part II of the Seeds Regulations under the Seeds Act specifies that seed potatoes must
be certified before they can be sold commercially (Department of Justice 2014). The seed
certification process ensures that the seed potatoes originated from nuclear stock, which means
that they were produced from sterile tissue culture propagules and subjected to a number of tests
that demonstrate it to be disease-free. The Canadian seed potato certification system relies on the
continuous introduction of disease free material in the seed supply continuum and limits
multiplication of seed potato stocks to a maximum of six field generations to minimize disease
build-up. Further disease and varietal purity standards are ensured through additional field
inspections, laboratory testing, post-harvest testing, and agronomic practices.

2.4 TAXONOMY AND GENETICS

Solanum tuberosum belongs to the Solanaceae family. This family includes, among 2000 other
species, tomato (S.lycopersicum L.), sweet pepper (Capsicum annuum L.), eggplant (S.
melongena L. var. esculentum), tobacco (Nicotiana tabacum L.), and petunia (Petunia hybrid L.).
The genus Solanum is a polymorphous and largely tropical and subtropical genus containing
more than 1000 species (Fernald 1970; Spooner and Knapp 2013).

Taxonomists have experienced difficulty in the ordering of species within the Solanum genus.
Species definitions are confounded by a number of factors, including similar morphologies
between distinct species, high levels of hybridization followed by introgression, and phenotypic
plasticity in variable environments (Spooner and Berg 1992). S. tuberosumhas been placed in the
section Petota, which includes all of the tuber-bearing wild and cultivated potatoes. It is also
informally classified in the Potato clade, which includes tomato and its wild relatives in section
Lycopersicon as well as the closely related section Etuberosum (Bohs 2005; Weese and Bohs
2007).

One of the more widely used classifications identified seven species of cultivated potato: S.
tuberosum, S. curtilobumJuz. & Bukasov, S. chaucha Juz. & Bukasov, S. juzepczukii Bukasov, S.
ajanhuiri, Juz & Bukasov, S. phureja Juz. & Bukasov, and S. stenotomum Juz. & Bukasov
(Hawkes 1990). However, this classification is not universally accepted. A recently updated
classification suggests there are only four cultivated species: S. tuberosum, S. ajanhuiri,
S.juzepczukii and S. curtilobum (Spooner et al. 2007).

Domestic S. tuberosum are highly heterozygous autotetraploids (2n = 4x = 48); however, some S.
spp. landraces cultivated primarily in South America are diploid (2n = 2x = 24), triploid (3x =
36), or pentaploid (5x = 60) (Andersson and de Vicente 2010). Continued self-pollination of S.
tuberosum can lead to large inbreeding depression due to the fact that many characteristics are
determined by non-additive genetic effects (Gopal and Ortiz 2006).

S. tuberosum is divided into two subspecies: tuberosum and andigena. The subspecies tuberosum
is the cultivated potato widely used as a crop in North America andEurope. The subspecies
andigena is also a cultivated species, but cultivation is restricted to Central and South America
(Hawkes 1990; OECD 1997).

Taxonomic position (USDA, NRCS, 2010):

Kingdom: Plantae (plants)

Subkingdom: Tracheobionta (vascular plants)

Superdivision: Spermatophyta (seed plants)

Division: Magnoliophyta (flowering plants)

Class: Magnoliopsida (dicotyledons)

Subclass: Asteridae

Order: Solanales

Family: Solanaceae

Subfamily: Solanoideae

Genus: Solanum L.

Section: Petota

Subsection: Potatoe

Series: Tuberosa

Species: Solanum tuberosum L.

2.5 HISTORY OF SYNTHETIC SEEDS

In general, there are two types of seeds which can be used for propagation of plants and thus help
in the maintaining the survival of plants in nature and these are Natural Seeds and Artificial
Seeds.

Natural Seed

The seed stage of seed plants represents a unique developmental phase of the spermatophyte life-
cycle, and as such involves structures, not characteristic of other stages of development. The
essential structure of seed is defined as a ripened ovule consisting of an embryo and its coat. The
normal seed contains materials which it utilizes during the process of its germination. There
substances are frequently found in the endosperm. Thus endosperm may contain variety of stored
materials such as starch, oils, proteins etc. In some plants, however, the reserve food material is
present in cotyledons.

Importance of natural seed

The seed provides an expedient living unit for the study of wholeness that is a complex of
biological factors which can be considered simultaneously. The seed occupies that sector of an
organism life cycle form mega sporogenesis (genetic) to the formation of seedling (ecological).
However, a seed is not truly a reproductive structure, but rather an adaptive mechanism to
facilitate suspending growth and interrupting the continuum of homeostasis in the life cycle.
Seeds are the corner stone of agriculture because when seeds are planted in the soil and given
water, nutrients, light and some protection from pests would reproduce plant and seeds identical
to that planted and also produce number of seeds which could be used for food or feed.

Synthetic seeds

Synthetic seeds are defined as artificially encapsulated somatic embryos, shoot buds, cell
aggregates, or any other tissue that can be used for sowing as a seed and that possess the ability
to convert into a plant under in vitro or ex vitro conditions and that retains this potential also
after storage. Earlier, synthetic seeds were referred only to the somatic embryos that were of
economic use in crop production and plant delivery to the field or greenhouses (Gray et al.,
1991). In the recent past, however, other micropropagules like shoot buds, shoot tips,
organogenic or embryogenic etc. Implementation of synthetic seed technology requires
manipulation of in vitro culture systems for large scale production of viable materials that are
able to convert into plants, for encapsulation, somatic embryogenesis, organogenesis and
enhanced auxiliary bud proliferation systems are the efficient techniques for rapid and large scale
in vitro multiplication of elite and desirable plant species. Through these systems a large number
of somatic embryos or shoot buds are produced which are used as efficient planting materials as
they are plant regeneration either after having minor treatment or without any treatment with
growth regulator(s). Because the unaired micropropagules are sensitive to desiccation and / or
pathogens when exposed to natural environment, it is envisaged that for large scale mechanical
planting and to improve the success of plant (in vitro derived) delivery to the field or greenhouse,
the somatic embryos or even the other micropropagules useful in synthetic seed production
would necessarily require some protective coatings. Encapsulation is expected to be the best
method to provide protection and to convert to in vitro derived propagules into synthetic seeds of
a number of plant species belonging to angiosperms and gymnosperms. Nevertheless, their
number is quite small in comparison to the total number of plant species in which in vitro
regeneration system has been established.

Technology

Basic hindrance to synthetic seed technology was primarily based on the fact that the somatic
embryos lack important accessory tissues, i.e., endosperm and protection coatings, that make
them in convenient to store and handle (Renden baugh, et al., 1993). Furthermore, they are
generally regarded to lack a quiescent resting phase and are incapable of undergoing
dehydration. The primary goal of synthetic seed research was, therefore, to produce somatic
embryos that resemble more closely the seed embryos in storage and handling characteristics so
that they can be utilized as a unit for clonal plant. Propagation and germplasm conservation. In
achieving such a goal, the technology of encapsulation has evolved as the first major step of
production of synthetic seeds. Later it was thought that the encapsulation synthetic seeds should
also contain growth nutrients, plant growth promoting micro-organisms (e.g., micorrhizae), and
other biological components necessary for optimal embryo-to plant development. The choice of
coating material for making synthetic seeds is also an important aspect for synthetic seed
production.
Based on the technology established so far, two types of synthetic seeds are known desiccated
and hydrated. The desiccated synthetic seeds are produced from somatic embryos either naked or
encapsulated in polyethylene glycol followed by their desiccation. Desiccation can be achieved
either slowly over a period of one or two weeks sequentially using chambers of decreasing
relative humidity or rapidly by unsealing the pier dishes and leaving then on the bench overnight
to dry. Such types of synthetic seeds are produced only in plant species whose somatic embryos
are desiccation tolerant. On the contrary, hydrated synthetic seeds are produced in those plants
where the somatic embryos are recalcitrant and sensitive to desiccation. Hydrated synthetic seed
are produced by encapsulating the somatic embryos are hydrogel capsules.

Synthetic seeds are defined as artificially encapsulated somatic embryos, shoot buds, cell
aggregates, or any other tissue that can be used for sowing as a seed and that possess the ability
to convert into a plant under in vitro or ex vitro conditions and that retain this potential also after
storage (Capuano et.al., 1998). Earlier, synthetic seeds were referred only to the somatic embryos
that were of economic use in crop production and plant delivery to the field or greenhouse (Gray
and Purohit, 1991; Janick, et.al., 1993). In the recent past, however, other micropropagules like
shoot buds, shoot tips, organogenic or embyrogenic calli, etc. have also been employed in the
production of synthetic seeds. Thus, the concept of synthetic seeds has been set free from its
bonds to somatic embryogenesis and links the term not only to its use (storage and sowing) and
product (plantlet) but also to other techniques of micropropagation like organogenesis and
enhanced axillary bud proliferation system. Implementation of synthetic seed technology
requires manipulation of in vitro culture systems for large-scale production of viable materials
that are able to convert into plants for encapsulation. Although results of intensive researches in
the field of synthetic seed technology seem promising for propagating a number of plant species,
practical implementation of the technology is constrained due to the following main reasons:

Limited production of viable micropropagules useful in synthetic seed production.

Anomalous and asynchronous development of somatic embryos.

Improper maturation of the somatic embryos that makes them inefficient for germination and
conversion into normal plants
Lack of dormancy and stress tolerance in somatic embryos that limit the storage of synthetic
seeds.

Poor conversion of even apparently normally matured somatic embryos and other
micropropagules into plantlets that limit the value of the synthetic seeds and ultimately the
technology itself.

The origin of the idea of an artificial seed is difficult to determine. Certainly, those who first
produced somatic embryos may have considered such application (Steward, et al., 1958 and
Reinert, 1958). The discovery of somatic embryogenesis in carrot in the year 1958 was almost
simultaneously by F. C. Steward (USA) and J. Reinert (Germany). F.C. Steward a renowned
plant physiologist at Cornell University in New York. However, it was not until the early 1970’s
that the concept of using somatic embryos began to be presented as a potential propagation
system for seed sown crops. Toshio Murashige gave a number of survivors in tissue culture
propagation where he concluded with this concept. He formally presented his ideas on artificial
seeds at the symposium on tissue culture for horticultural purposes in Belgium, September 6-9,
1977. His terse comments in the proceedings, however, were to be applicable, the cloning
method must be extremely rapid, capable of generating several million plants daily and
competitive economically with the seed method (Mugashige, 1977). Drew (1979) was active in
developing methods to commercially propagate crops using somatic embryos. He suggested
delivering carrot somatic embryos in a fluid drilling system, but was able to produce only three
plants from carrot embryos on a carbohydrate free medium. He could not get success in
producing many plants through this system. He faced a crucial problem and found the very slow
rate of development of plantlets derived from culture. Kitto and Janick (1982) coated dumps of
carrot embryos, roots and Callus with polyethylene. Some embryos survived the coating process
as well as a desiccation step (Kitto and Janick, 1985a and 1985b). The early assessment of
Murashige (1977) on the difficulty of somatic embryos are still valid today. The quality and
fidelity of somatic embryos are the limiting factors for development and scale up of artificial
seeds.
CHAPTER 3: MATERIALS AND METHODS

3.1 Sterilisation of explants

Collect the immature part of shoots and nodal segments where the auxiliary bud is to be collected
from on the young plants of Solanum Tuberosum raised in the greenhouse for the production of
in vitro explants. Surface sterilize with 0.1% (w/v) HgCl 2 for 3 min and wash thoroughly with
sterile distilled water.

3.2 Production of Micro Shoots and auxiliary bud

Use micro shoots and auxiliary buds obtained when two different forms of explants when
cultured on MS medium supplemented with 1.0-3.0 mg/l BAP for 6 weeks on culture media.
Excise micro shoots sizes ranging from 3.0-5.0 mm from the explants and prepare them for
encapsulated to form artificial seed.

3.3 Preparation of MS basal Medium

Prepare MS basal medium using MS stock solution at pH 5.8. Rinse artificial seeds from the
previous step using this solution. Autoclave MS basal medium for 21 minutes at 121 oC prior, to
obtaining sterile solution.

3.4 Preparation of Sodium Alginate Solution (NaC6H7O6)

In this experiment, sodium alginate that consists of agonic acid, which is a type of sodium salt is
to be used as the encapsulation matrix. Prepare Sodium alginate solutions at 1.0%, 2.0%, 3.0%,
4.0% and 5.0% concentrations using a heating method by Fabre and Deridder (1990). Sodium
alginate solution is to be prepared in 100ml MS basal medium without the additions of calcium
chloride dehydrate (CaCl2.2H20). To prepare 1.0% sodium alginate solution, 1.0 g of sodium
alginate powder is added slowly in MS basal medium without calcium chloride dehydrate
(CaCl2.2H20) to allow it to dissolve completely and followed by the addition of 3.0 g sucrose,
then autoclave for 21 minutes at 121 oC. Sterilize the sodium alginate solution to be used as an
encapsulation matrix for the production of artificial seeds.

3.5 Preparation of Calcium Chloride Dehydrate Solution

Calcium chloride dehydrate (CaCl2.2H2O) acts as complexing solution that allows the hardening
of encapsulated matrix. Prepare different concentrations of CaCl 2.2H2O (25-125 mm) in liquid
MS medium. To prepare 25mM CaCl 2.2H2O solution, 0.9189 g of CaCl2.2H2O powder is added
into 250 ml MS liquid basal medium. Autoclave this solution for 21 minutes at 121 oC.

3.5 Sodium Alginate Solution (NaC6H7O6) and Calcium Chloride Dehydrate Solution in
Different Concentrations

Prepare five different concentrations of sodium alginate solution at 1.0%, 2.0%, 3.0%, 4.0% and
5.0% respectively and also five different concentrations of CaCl 2.2H2O prepared at 25 mm, 50
mm, 75 mm, 100 mm and 125 Mm respectively. The encapsulation matrix will be tested using
all five concentrations of sodium alginate solution. The beads formed are to be soaked in
CaCl2.2H2O at different concentrations for 30 minutes. Results are to be obtained based on the
observations on the shape of beads formed. Thirty replicates are to be used in each treatment.
From this experiment the optimum sodium alginate and calcium chloride dehydrate solution to
form the ideal beads is to be identified. Synthetic seeds formed are to be germinated on MS solid
and liquid media.

3.6 Preparation of Encapsulation Matrix in Different Solution

Encapsulation matrix is to be prepared by dissolving sodium alginate powder in MS basal liquid

medium (without CaCl2.2H2O) with the addition of sucrose. In this experiment, sodium alginate
powder will be dissolved in 4 different solutions which are;

MS basal liquid medium

MS basal liquid medium (without CaCl2.2H2O)

MS basal liquid medium (without CaCl 2.2H2O) added with 2.0 mg/l BAP + 0.5 mg/l NAA
(optimum hormone for regeneration of shoots)

Distilled water

Four different types of encapsulation matrix are to be prepared using these solutions. Three
percent of sodium alginate and 3.0 g sucrose are to be dissolved in each of the solution. Micro
shoots and auxiliary buds are then encapsulated using the encapsulation technique. Beads are to
be cultured and germinated on MS basal solid medium in the culture room at 25 ± 1 oC and 16
hours’ light and 8 hours’ dark. Observations are to be made daily and all results to be recorded.
Thirty replicates are to be used in each treatment.

3.7 Germination of Synthetic Seeds on Various Sowing Media

The success of the production of synthetic seeds is to be determined by absorbing and recording
the ability of the seeds to germinate, grow and finally acclimatize and develop into normal
plants. Synthetic seeds formed from the encapsulation of micro shoots composed of Ca-free MS
with the addition of 3.0 % sodium alginate, 3.0 % sucrose and supplemented with 2.0 mg/l BAP
and 0.5 mg/l NAA will be germinated on 3 different types of sowing media. MS basal liquid
medium, sterile garden soil and sterile vermiculite will be used in this experiment. The most
suitable sowing medium will be identified.

3.8 Data Analysis

All in vitro experiments designed according to the Complete Randomized Design (CRD)
Statistical analysis will be done using IBM SPSS Statistics 20 software. Mean separation will
have done by Duncan Multiple range test, Dennett’s test and Fisher’s exact test at the confidence
interval of 95%.
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