Artificial seed & and their application

SEMINAR-1
ON
ARTIFICIAL SEED AND THEIR
APPLICATIONS.
ABHINASH MOIRANGTHEM
PGS09AGR5084

INTRODUCTION
WHAT IS ARTIFICIAL SEED..?
ARTIFICIAL SEED CAN BE DEFINED AS ARTIFICIAL
ENCAPSULATION OF SOMATIC EMBRYOS, SHOOT
BUD OR AGGREGATES OF CELL OF ANY TISSUES
WHICH HAS THE ABILITY TO FORM A PLANT IN
IN-VITRO OR EX-VIVO CONDITION. ARTIFICIAL
SEED HAVE ALSO BEEN OFTEN REFERRED TO AS
SYNTHETIC SEED.

ARTIFICIAL SEEDS
Concepts -
• Artificial seeds were first introduced in 1970’s as a novel
analogue to the plant seeds. The production of artificial
seeds is useful for plants which do not produce viable
seeds. It represents a method to propagate these plants.
Artificial seeds are small sized and these provides
further advantages in storage, handling and shipping.
• The term, “EMBLING” is used for the plants originated
from synthetic seed.
• The use of synthetic varieties for commercial cultivation
was first suggested in Maize (Hays & Garber, 1919).

ARTIFICIAL SEED
SOMATIC EMBRYO
ARTIFICIAL
ENDOSPERM
THE CONCEPT OF ARTIFICIAL SEED

BASED ON THE TECHNIQUES TWO TYPES OF
ARTIFICIAL SEEDS ARE PRODUCED
• DESICCATED SYNTHETIC SEEDS- Desiccated synthetic seeds
are produced nacked or polyoxyethylene glycol encapsulated
somatic embryos. This type of synthetic seeds is produced in
desiccation tolerant species plant.
• HYDRATED SYNTHETIC SEEDS- Hydrated synthetic seeds
are produced by encapsulating the somatic embryos in hydrogels
like sodium alginate, potassium alginate, carrageenan, sodium
pectate or sodium alginate with gelatine.

NEED FOR ARTIFICIAL PRODUCTION
TECHNOLOGY
 Development of micro propagation technique will
ensure abundant supply of desired plant species.
 Development of artificial seed production technology
is currently considered as an effective and efficient
method of propagation in several commercially
important agronomic and horticultural crops.

 These artificial seed would also be a channel for new
plant lines produced through biotechnological advances
to be delivered directly to greenhouse and field.
 High volume propagation potential of somatic embryos
combined with formation of synthetic seeds for low-cost
delivery would open new vistas for clonal propagation in
several commercially important crop species.

WHAT ARE SOMATIC EMBRYOS ?
Somatic embryos are bipolar structures
with both apical and basal meristematic
regions, which are capable of forming
shoot and root respectively.

SOMATIC EMBRYOS vs ZYGOTIC EMBRYOS AND
THEIR ADVANTAGES.
 Somatic embryos are structurally similar to zygotic
embryos found in seeds and possess many of their
useful features, including the ability to grow into
complete plant.
 Somatic embryos differ in that they develop from somatic
cells, instead of zygotes and thus, potentially can be
used to produces duplicates of single genotypes.

 Somatic embryos develops from somatic cells(non-
sexual) and does not involve sexual recombination. This
characteristic of somatic embryos allows not only clonal
propagation but also specific and directed changes to be
introduced into desirable elite individuals by inserting
isolated gene sequences into somatic cells.
 If the production efficiency and convenience comparable
to that of a true seed are achieved, somatic embryos can
be potentially used as a clonal propagation system.

BASIC REQUIREMENT FOR THE PRODUCTION OF
ARTIFICIAL SEEDS.
 One pre-requisite for the application of synthetic seed
technology in micropropagation is the production of high
quality, vigorous somatic embryos that can produce
plants with frequencies comparable to natural seeds.
 Synthetic seed technology requires the inexpensive
production of large numbers of high quality somatic
embryos with synchronous maturation.

 Encapsulation and coating systems, though important for
delivery of somatic embryos, are not the limiting factors
for the development of synthetic seeds.
 The lack of synchrony of somatic embryos is, arguably,
the single most important hurdle to be overcome before
advances leading to wide spread commercialization of
synthetic seeds can occur.

PROCEDURE FOR PRODUCTION OF ARTIFICIAL SEEDS
Establish somatic embryogenesis
Mature somatic embryogenesis
Synchronize and singulate somatic embryos
Mass production of somatic embryos
Standardization of encapsulation
Standardization of artificial endosperm
Mass production of artificial seeds
Greenhouse and field planting

Methods for artificial seed encapsulation
• Dropping method
Somatic embryos are dipped in hydrogel, this step
encapsulate SEs.
Hydrogel used may be any of the following.
o alginate – sodium alginate, agar from see weeds, seed gums
like guar gum, locust bean gum.
- Sodium alginate solution (1 – 5%), prepared in MS basal medium
solution.
- SEs are dipped in this solution.
-These coated beads are added one by one into a complexation
solution flask kept on magnetic stirrer and kept such for around
20-30 minutes.

o Embryos get covered by calcium alginate which is a stable
complex due to ionic bond formation, become harder, Seeds
become harder.
o Then gelled embryos are washed with water or MS basal
medium.
o The synthetic seeds are ready.
Molding method
• This method follows simple procedure of mixing of embryos
with temperature dependent gel (e.g. gel rite, agar).
• Cells get coated with the gel at lowering of the temperature.

A. Encapsulated shoot tips in sodium alginate bead.
B. Germination of encapsulated shoot tips.
C. Shoot induction of encapsulated shoot tips.
D. Shoots from encapsulated shoot tips
E. Rooted multiple shoots of encapsulated shoot tips.
F. Established plant in earthen pot.
Fig: Regeneration of encapsulated shoot tips (artificial seed) of pointed gourd.

THE ALFA ALFA TISSUE CULTURE SYSTEM USED
TO PRODUCED SYNTHETIC SEED

THE MORPHOLOGICAL STAGES OF SOMATIC EMBRYO
DEVELOPMENT IN ALFA ALFA (Medicago sativa L.)

Figure - Protocorm-like bodies (PLBs), encapsulated
PLBs and their regeneration under in vitro and natural
condition.
(A) PLBs selected for encapsulation. (B) Encapsulated PLBs.
(C) In vitro regeneration of PLBs. (D) Regeneration and emergence of artificial
seeds under in vivo conditions

DESICCATION TOLERANCE:
 Desiccation tolerance is a quantitative characteristic not
a qualitative one.
 It can be induced by a pretreatment with ABA or stress
to elicit the desired response.
 The type of pretreatment used, the duration for which it
is applied and the stage of embryo that is treated are
critical factors.
 For 3 days in 20 mm ABA is sufficient to induce
tolerance, but chilling requires almost 3 weeks.

OVERVIEW OF DRY SYNTHETIC SEED
PRODUCTION:
1. After pollination, a zygotic embryo of a dicotyledonous
species develops through a series of morphological
stages termed globular, heart and torpedo.
2. Cotyledons develop and expand as the storage reserves
of protein, starch and/or oil are deposited.
3. Before the embryo achieves its maximum weight, it
acquires the ability to tolerate drying.
4. Then, the seed's vascular connections to the maternal
plant are severed, it stops importing nutrients and it
begins to lose water. Once they are hydrated,
germination commences culminating in the emergence
of a radicle and then the mobilization of the storage
reserves by the seedling.

SCHEMATIC REPRESENTATION OF THE MAJOR STAGES IN
ZYGOTIC EMBRYO DEVELOPMENT
FROM POLLINATION TO GERMINATION

TYPES OF GELLING AGENTS USED FOR
ENCAPSULATION
• Several gels like agar, alginate, polyco2133 (Bordon Co.),
carboxy methyl cellulose, carrageenan, gelrite (Kelko. Co.),
guargum, sodium pectate, tragacanth gum, etc. Were
tested for synthetic seed production, out of which alginate
encapsulation was found to be more suitable and
practicable for synthetic seed production.
• Alginate was chosen because it enhances capsule
formation and also rigidity of alginate beads provides better
protection to the encased somatic embryos against
mechanical injury.

PRINCIPLE AND CONDITIONS FOR ENCAPSULATION
WITH ALGINATE MATRIX
• The major principle involved in alginate encapsulation
process is that the sodium alginate droplets containing
the somatic embryos when dropped into CaCl2.2H2O
solution form round and firm beads due to ion exchange
between the NA+
in sodium alginate with CA2+
in the
CaCl2.2H2O solution.
• 3% sodium alginate upon complexation with 75mM
CaCl2.2H2O for half an hour gives optimum beads
hardness and rigidity for the production of viable
synthetic seeds.

ARTIFICIAL ENDOSPERM
 Somatic embryos lack seed coat (testa) and endosperm
that provide protection and nutrition for zygotic embryos
in developing seeds.
 To augment these deficiencies, addition of nutrients and
growth regulators to the encapsulation matrix is desired,
which serves as an artificial endosperm.
 These addition results in increase efficiency of
germination and viability of encapsulated somatic
embryos.these synthetic seeds can be stored for a
longer period of time even upto 6 months without losing
viabilty,especially when stored at 40
c.

ADDITION OF ADJUVANTS TO THE MATRIX
To prevent the embryo from desiccation and mechanical
injury, a number of useful materials such as nutrients,
fungicides, pesticides, antibiotics and microorganisms
(eg.rhizobia) may be incorporated into the encapsulation
matrix.
Incorporation of activated charcoal improves the
conversion and vigour of the encapsulated somatic
embryos and retains nutrients within the hydrogel
capsule and slowly releases them to the growing
embryo.

Additives Number of multiple shoots produced in diff. dendrobium hybrids
D.Kasem
Gold X
D.Thed
Takiguchi
D.sonia D.New
Sabin
Red
D.Ekapol
Panda
No. 1
D.Sakura
Pink
D.Banyad
Pink
CW (20%)+NAA (0.1)+BAP (2) 5 4 8 6 3 4
CW (20%)+NAA (0.1)+BAP (3) 8 6 12 7 5 4
NAA (0.1) + BAP (2) 3 3 5 2 2 3
NAA (0.1) + BAP (3) 2 3 6 4 4 4
MULTIPLE SHOOT FORMATION IN SHOOT TIP EXPLANTS FO DIFFERENT
DENDROBIUM HYBRIDS IN VW MEDIUM CONTAINING DIFF.GROWTH ADJUNCTS
CAU, Imphal Devi et al., (1998)

Effect of different concentrations of sodium bicarbonate on in vivo germination of
G.densiflorum. Each set consists of 25 encapsulated PLBs and had 10 replicates.
Concentration of sodium
bicarbonate (mg1-1
)
Concentration of
bavastin (mg1-1
)
Regeneration
percentage ± SE
0 4 6.00±0.30
5 4 12.00±1.08
10 4 20.4**±1.69
15 4 24.2**±1.28
20 4 28.40**±1.04
25 4 22.6*±0.60
30 4 20.20**±0.74
40 4 18.6***±0.78
Germination percentage followed by asterisks in each treatment within
the same column is significantly different from control (artificial seeds
without NaHCO3) using student’s t test at *5% level; **1% level and
***0.1% level.
RAMMOHANPUR (WB) DATTA et al., (1999)

UTILIZATION OF ARTIFICIAL SEEDS
 The artificial seeds can be used for specific purposes,
notably multiplication of non-seed producing plants,
ornamental hybrids or the propagation of polyploid plants
with elite traits.
 It can be employed in the propagation of male or female
sterile plants for hybrid seed production.
 Cryo-preserved artificial seeds may also be used for
germplasm preservation particularly in recalcitrant
species (such as mango, cocoa and coconut), as these
seed will not undergo desiccation.

 Transgenic plants, which require separate growth
facilities to maintain original genotypes may also be
preserved using somatic embryos.
 Somatic embryogenesis is a potential tool in the genetic
engineering of the plants.
 Plants that are generated by somatic embryos from
single transgenic cell, the progeny will not be chimeric.

 Multiplication of elite plants selected in plant breeding
programs via somatic embryos avoids the genetic
recombination, and therefore does not warrant continued
selection inherent in conventional plant breeding, saving
considerable amount of time and other resources.
 Artificial seeds produced in tissue culture are free of
pathogens. Thus, another advantage is the transport of
pathogen free propagules across the international
borders avoiding bulk transportation of plants, quarantine
and spread of diseases.

POTENTIAL USES OF ARTIFICIAL SEEDS
DELIVERY SYSTEMS:
 Reduced costs of transplants.
 Direct greenhouse and field delivery of:
- elite, select genotypes
- hand-pollinated hybrids
- genetically engineered plants
- sterile and unstable genotypes

 Large-scale mono cultures.
 Mixed-genotype plantations.
 Carriers for adjuvants such as microorganisms,
plant growth regulators, pesticides, fungicides,
nutrients and antibiotics.
 Protection of meiotically-unstable, elite genotypes.
 Can be conceivably handled as seed using
conventional planting equipment.

ANALYTICAL TOOLS:
 Comparative aid for zygotic embryogeny.
 Production of large numbers of identical embryos.
 Determination of role of endosperm in embryo
development and germination.
 Study of seed coat formation.
 Study of somoclonal variation.

APPLICABILITY AND FEASIBILITY OF ARTIFICIAL
SEED PRODUCTION TECHNOLOGY
 In order to be useful, synthetic seed must either reduce
production costs or increase crop value.
 The relative benefits gained, when weighed against
development costs, will determine whether its use is
justified for a given crop species.
 For e.g. synthetic seed of seedless water melon would
actually cost less than conventional seed, providing a
benefit at the outset of crop production.

Artificial seed & and their application

Artificial seed & and their application

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