banana tissue culture and synthetic seeds
TRANSCRIPT
Banana tissue culture and synthetic seeds:
Banana is a globally important fruit crop with 97.5 million tones of production. In India it
supports livelihood of millions of people. With total annual production of 16.91 million tons
from 490.70 thousand ha. with national average of 33.5 T/ha. Maharashtra ranks first in
production with 60 T/ha. Banana contributes 37% to total fruit production in India.
Banana is one of the major and economically important fruit crops of Maharashtra. Banana
occupies 20% area among the total area under crop in India. Maharashtra ranks second in
area and first in productivity in India. Jalgaon is a major Banana growing district in
Maharashtra which occupies 50,000 hectares area under Banana. But most of Banana is
grown by planting suckers. The technology development in agriculture is very fast, it results
in developing Tissue Culture Technique.
Tissue culture means cloning and micro-propagation of tissues of the selected Elite plants and
daughter suckers. The process consists of five important steps: Initiation, Multiplication,
Shooting & rooting, Primary Hardening in green houses and Secondary Hardening in shade
houses. Strict adherence to aseptic standards and micro-climatic conditions and care during
the hardening process alone can ensure success.
Banana is basically a tropical crop, grows well in temperature range of 13ºC – 38ºC with RH
regime of 75-85%. In India this crop is being cultivated in climate ranging from humid
tropical to dry mild subtropics through selection of appropriate varieties like Grandnaine.
Chilling injury occurs at temperatures below 12ºC. The normal growth of the banana begins
at 18ºC, reaches optimum at 27ºC, then declines and comes to a halt at 38ºC. Higher
temperature causes sun scorching. High velocity wind which exceeds 80 km phrs damages
the crop. Soil for banana should have good drainage, adequate fertility and moisture. Deep,
rich loamy soil with pH between 6-7.5 are most preferred for banana cultivation. Ill drained,
poorly aerated and nutritionally deficient soils are not suitable for banana. Saline solid,
calcareous soil are not suitable for Banana cultivation. Avoided soil of low laying areas, very
sandy & heavy black cotton with ill drainage.
Plant tissue culture comprises a set of in vitro techniques, methods and strategies that are part
of the group of technologies called plant biotechnology. Tissue culture has been exploited to
create genetic variability from which crop plants can be improved, to improve the state of
health of the planted material and to increase the number of desirable germplasms available
to the plant breeder. Tissue-culture protocols are available for most crop species, although
continued optimization is still required for many crops, especially cereals and woody plants.
Tissue culture techniques, in combination with molecular techniques, have been successfully
used to incorporate specific traits through gene transfer. In vitro techniques for the culture of
protoplasts, anthers, microspores, ovules and embryos have been used to create new genetic
variation in the breeding lines, often via haploid production. Cell culture has also produced
somaclonal and gametoclonal variants with crop-improvement potential. The culture of single
cells and meristems can be effectively used to eradicate pathogens from planting material and
thereby dramatically improve the yield of established cultivars. Large-scale micropropagation
laboratories are providing millions of plants for the commercial ornamental market and the
agricultural, clonally-propagated crop market. With selected laboratory material typically
taking one or two decades to reach the commercial market through plant breeding, this
technology can be expected to have an ever increasing impact on crop improvement as we
approach the new millenium.
A soil that is not too acidic & not too alkaline, rich in organic material with high nitrogen
content, adequate phosphorus level and plenty of potash are good for banana.
Prior to planting banana, grow the green manuring crop like daincha, cowpea etc. and burry it
in the soil. The land can be ploughed 2-4 times and leveled. Use ratovator or harrow to break
the clod and bring the soil to a fine tilt. During soil preparation basal dose of FYM is added
and thoroughly mixed into the soil.
A pit size of 45cm x 45cm x 45cm is normally required. The pits are to be refilled with
topsoil mixed with 10 kg of FYM (well decomposed), 250 gm of Neem cake and 20 gm of
conbofuron. Prepared pits are left to solar radiation helps in killing the harmful insects, is
effective against soil borne diseases and aids aeration. In saline alkali soil where PH is above
8 Pit mixture is to be modified to incorporate organic matter.
Addition of organic matter helps in reducing salinity while addition of purlite improves,
porosity and aeration. Alternative to planting in pits is planting in furrows. Depnding on soil
strata one can choose appropriate method as well as spacing and depth at which plant is
required to be planted.
Sword suckers weighing approximately 500-1000 gm are commonly used as propagating
material. Suckers generally may be infected with some pathogens and nematodes. Similarly
due to the variation in age and size of sucker the crop is not uniform, harvesting is prolonged
and management becomes difficult.
Therefore, in-vitro clonal propagation i.e. Tissue culture plants are recommended for
planting. They are healthy, disease free, uniform and authentic. Properly hardened secondary
seedlings are only recommended for planting
Planting of tissue culture Banana can be done throughout the year except when the
temperature is too low or too high. Facility of drip irrigation system is important. There are
two important seasons in Maharashtra, India;
Mrig Baug (Kharif) Month of planting June - July.
Kande Baug (Rabi) Month of planting October - November.
Polybags is separated from the plant without disturbing the root ball of the plant and then
plants are planted in the pits keeping the pseudo-stem 2cm below the ground level. Soil
around the plant is gently pressed. Deep planting should be avoided.
Banana, a water loving plant, requires a large quantity of water for maximum productivity.
But Banana roots are poor withdrawal of water. Therefore under Indian condition banana
production should be supported by an efficient irrigation system like drip irrigation.
Water requirement of banana has been worked out to be 2000mm per Annum. Application of
drip irrigation and mulching technology has reported improved water use efficiency. There is
saving of 56% of water and increasing yield by 23-32% under drip.
Irrigate the plants immediately after planting. Apply sufficient water and maintain field
capacity. Excess irrigation will lead to root zone congestion due to removal of air from soil
pores, thereby affecting plant establishment and growth. And hence drip method is must for
proper water management in Banana
Month (Maug
Baug)
Qty.
(lpd.)
Month (Kande
baug)
Qty.
(lpd.)
June 06 October 04-06
July 05 November 04
August 06 December 04
September 08 January 06
October 10-12 February 08-10
November 10 March 10-12
December 10 April 16-18
January 10 May 18-20
February 12 June 12
March 16-18 July 12
April 20-22 August 14
May 25-30 September 14-16
Banana requires high amount of nutrients, which are often supplied only in part by the soil.
Nutrient requirement has been worked out on all India basis is to be 20 kg FYM, 200gm N;
60-70gm P; 300gm K/plant. Banana requires heavy nutrition. Banana crop requires 7-8 Kg N,
0.7- 1.5 Kg P and 17-20 Kg K per metric tonne yield. Banana responds well to application of
nutrients. Traditionally farmers use more of urea and less of phosphorous and potash.
In order to avoid loss of nutrients from conventional fertilizers i.e. loss of N through leaching,
volatilization, evaporation and loss of P and K by fixation in the soil, application of water
soluble or liquid fertilizers through drip irrigation (fertigation) is encouraged. A 25-30%
increase in yield is observed using fertigation. Moreover, it saves labour and time and the
distribution of nutrients is uniform
Water Soluble Solid fertilizers
Schedule of water soluble fertilizer application.
Period Grade
Qty. per 1000
plants (Kg)
every 4th day
basis
Total Qty.
(Kg.)
After planting upto
65 days
Urea 4.13 82.60
12:61:003.00 60.00
00:00:505.00 100.00
65 to 135 days
Urea 6.00 120.00
12:61:002.00 40.00
00:00:505.00 100.00
135 to 165 daysUrea 6.50 65.00
00:00:506.00 60.00
165 to 315 daysUrea 3.00 150.00
00:00:506.00 300.00
Schedule is directive only and may change according to planting season and soil fertility
status (soil analysis).
Interculture Operations
The Root system of banana is superficial and easily damaged by cultivation, use of intercrop
which is not desirable. However short durational crops (45-60 days) like mung, cowpea,
daincha are to be considered as green manuring crops. Crops from cucurbitaceous family
should be avoided as these carry viruses.
Weeding
Spraying of Glyphosate (Round up) before planting at the rate of 2 lit/ha is carried out to
keep the plantation weed free. One or two manual weedings are necessary.
Micronutrient Foliar Spray
Combined foliar application of ZnSo4 (0.5%), FcSo4 (0.2%), CuSo4 (0.2%) and H3Bo3
(0.1%) can be adopted to improve morphological, physiological and yield attributes of
banana. The micronutrient spray solution is prepared by dissolving the following in 100 lit. of
water.
Zinc
sulphate
500
gm
For every 10 litre of mixture 5-
10ml of sticker solution such as
Teepol should be added before
spraying.
Ferrom
sulphate
200
gm
Copper
sulphate
200
gm
Boric acid100
gm
Desuckering
Removal of unwanted suckers is a critical operation in banana for reducing internal
competition with mother plant.
Desuckering should be done regularly until shooting. However in areas where ratoon is also
taken for the second crop, a follower is allowed after inflorescence has appeared and this
should be managed that planting space is not disturbed. Follower should be opposite to the
inflorescence. It should not be far apart from the main plant.
Deflowering
It consists of removal of the withered style and perianth. This is generally not practiced.
Therefore, they remain attached to the fruit bunch & then removed after harvesting which is
damaging to the fruits. It is therefore suggested that you remove them just after flowering.
Pruning of leaves
Rubbing leaves damages the fruit, therefore, such leaves should also be pruned during regular
check. Older leaves and infected leaves also be pruned as required. Green leaves should not
be removed.
Earthing up
Keep the soil loose by harrowing from time to time. Earthing up should be done at 3-4
months after planting i.e. raising the soil level around the base of the plant by 10-12”. It is
better to prepare a raised bed and keep the drip line on bed 2-3” away from the plant. It also
helps to protect plants from wind damage and production losses to some extent..
Removal of male buds
(Denavelling) Removal of male buds helps fruit development and increases bunch weight.
Male buds are removed from the last 1-2 small hands with a clean cut keeping a single finger
in the last hand.
Bunch Spray
Spray of monocrotophos (0.2%) after emergence of all hands takes care of the thrips. Thrips
attack discolors the fruit skin and makes it unattractive.
Bunch Covering
Covering bunch using dried leaves of the plant is economical and prevents the bunch from
direct exposure to sunlight. Bunch cover enhances quality of fruit. But in rainy season this
practice should be avoided.
Sleeving of bunch is done to protect fruits against dust, spray residue, insect and birds. For
this blue plastic sleeves are preferred. This also increases temperature around developing
bunch and helps in early maturity.
Dehandling of false hands of bunch
In a bunch there are some incomplete hands which are not fit for quality produce. These
hands should be removed soon after bloom. This helps in improving the weight of other
hands. Sometimes the hand just above the false hand is also removed.
Propping
Due to heavy weight of bunch the plant goes out of balance and the bearing plant may lodge
and production and quality are adversely affected. Therefore they should be propped with the
help of two bamboos forming a triangle by placing them against the stems on the leaning
side. This also helps in uniform development of bunch.
Pest and disease management
A large number of fungal, viral and bacterial diseases and insect pests and nematodes infest
the banana crop and reduce production, productivity and quality. Summary details of major
pest and diseases of banana along with control measures are given herewith:
Sno.Name Symptoms Control
measures
Pest
i) Rhizome weevil
(Cosmopolites
Sordidus)
a) Large
creates
network of
galleries in
rhizome and
weakens the
plant.
a) Use healthy
planting
material
b) Sanitation in
orchard
c) Trapping of
adult weevils
using
pseudostem or
rhizome pieces
and
d) Soil
application of
carbufuran
@.2gm/plant
ii) Pseudostem weevil
(Odaiporous
longicolis)
a) Small holes
on
pseudostem
with
exudation of
transparent
gummy
substance
a) Management
approach is
identical to
rhizome weevil
b) Existence
tunneling in
leaf sheath
and inner core
of the stem
b) Secondly,
injection of lime
solution
(Monocrotophos
150 ml in 350
ml water) using
stem injector 4
ft. above the
ground level at
30º angle is
recommended.
c) Abortion of
bunches
c) Use
longitudinal
split (30cm
length) or disc
on stump traps
@ 100/ha. Keep
the split portion
of tray facing
the ground.
Collected
weevils are then
killed.
iii) Thrips
(Chaetanaphotrips
& signipennis &
Heliaothrips
kodaliphilus)
a) They scrap
from attacked
plant organs
and render
them brown
and discolored
especially the
fruits.
a) Spray or
inject
Monocrotophos
@ 0.05% on the
inflorescence
before the
unfurling of top
most bract.
iv) Fruit scarring battle
(Besilepta
subcostatum)
a) Adults feed
on tender
unfolded
leaves and
a) Sanitation
spray of 0.05%
moncrotophos
or 0.1%
fruits and
cause scarring
of skin
carbaryl on the
heart of the
plants
immediately
after the
emergence of
new foliage and
during fruiting
season is
recommended.
b) Plant losses
its vigour and
quality of
bunch is poor)
v) Aphids (Pentalonia
nigronervosa)
a) They are
vecturs of
banana
bunchy top
visus (BBTV)
and can be
seen as
congregation
under the leaf
base of
pseudo stem
a) Spray of
0.1%
monocrotophos
or 0.03%
phosphonidon
on the leaves is
effective
vi) Nematodes a) Stunted
growth
a) Apply
corbofuron @40
gm per plant at
planting & 4
month after
planting.
b) Small
leaves
c) Cutted
roots
b) Use neem
cake as organic
manure.
d) Purple
black lesions
on roots and
their splitting.
c) Use merigold
as trap crop.
Fungal Diseases
vii) Panama wilt
(Furarium
oxysporium)
a) Yellowing
of old leaves
progressing
a) Cultivation of
resistant cultivar
towards
younger leaves.
(Covendish
group)
b) Affected
leaves
collapse near
petiole and
hang.
b) Trim and
treat the suckers
in 0.1%
Bavistine before
plant.
c) Pseudo
stem splitting
is common.
c) Apply
bioagents like
trichoderma and
Pseudomonas
fluorescence
with organic
manure
d) Reddish
brown
discoloration
in cross-
section of root
& rhizome
d) Keep good
drainage and
apply lot of
organic manure
in field.
viii Head rot (Erwinia
carotovora)
a) Rotting of
collar region
and epinasty
of leaves)
a) Use healthy
planting
material
b) On pulling
out of affected
plant, the
plant topples
from the
collar region
leaving the
corn with root
in soil
c) On opening
up of collar
region of
affected
plants,
yellowish to
reddish ooze
can be seen.
b) Drench plants
with 0.1%
Emison
followed by
another
drenching after
3 months.
d) In early
stage of
infection, dark
c) Avoid
planting in
rocks and in
brown or
yellow, water
soaked areas
in critical
region which
may decay to
form cavities
surrounded by
dark spongy
tissues.
poorly drained
soils.
ix) Sigatoka leaf spot
(Mycospharella
spp)
a) It is
characterized
by small
lesions on the
leaves, the
lesion become
pale yellow to
greenish
yellow streaks
visible from
both the
surfaces of
leaf
a) Remove
infected leaves
and destroy
b) Thereafter
linear
brownish to
blackish
streaks
appear.
c) The centre b) Keep proper
of the streak
eventually
dries up and
give
appearance of
eye spot.
drainage and
avoid water
logging.
d) Some times
premature
ripening is
observed
c) Spray dithane
M-45 (1250
g/ha) or
Bavistine 500
g/ha.
Viral Diseases
i) Banana Bunchy
Top Virus(BBTV)
a) Appearance
of irregular,
dark green
'Morse code'
streaks along
secondary
veins on
leaves on
underside of
the leaves.
a) Use virus
free planting
material i.e.
Tissue Culture.
b) Survey and
eradicate
infected plants
regularly.
b) Leaf size is
reduced and
leaves remains
abnormally
erect, brittle
and results.
c) Control insect
vectors
especially
aphids and
mealy bugs.
d) Indexing
should be
followed in the
case of mass
multiplication
c) Leaves
short, close to
each other,
and bunched
at the top
e) Prohibit
movement of
any plant part
from diseased
area to healthy
area.
d) The tips of
the bracts in
male buds
have greenish.
f) Use resistant
cultivar.
e) Virus is
spread
through
aphids.
g) Avoid
growing of
alternate lost as
mixed crop or in
near by areas.
ii) Banana Mosaic
Virus (BMV)
a) Chlorosis
with mild
chlorotic
streaks along
the veins they
never turn
necrotic as in
BSV.
a) Elimination
of affected
plants and
maintenance of
disease free
plantation
through the use
of disease free
planting
material i.e.
Tissue culture
seeding.
iii) Banana Bract a) Presence of a) Use of
Mosaic Virus
(BBMV)
spindle shaped
pinkish to
reddish
streaks on
pseudo stem,
mid ribs,
petioles and
lamina.
disease free
planting
material i.e.
Tissue culture
seeding.
iv) Banana Streak
Virus (BSV)
a) Presence of
inconspicuous
chlorotic
flecking to
small lethal
systematic
necrosis, and
includes
yellow, brown
and black
streaking,
cigar leaf
necrosis,
based pseudo
stem splitting
internal
internal
pseudo stem
necrosis and
formation of
small
deformed
a) Use of
disease free
planting
material i.e.
Tissue culture
seedings.
bunches.
Harvesting
Banana should be harvested at the physiological maturity stage for better post harvest quality.
The fruit is climacteric and can reach consumption stage after ripening operation
Maturity indices
These are established on the basis of fruit shape, angularity, grade or diameter of the median
figure of the second hand, starch content and number of days that have elapsed after
flowering. Market preferences can also affect the decision for harvesting a slight or full
mature fruit.
Removal of bunch
Bunch should be harvested when figures of second hand from top are 3/4 rounded with the
help of sharp sickle 30cm above the first hand. Harvest may be delayed upto 100-110 days
after opening of the first hand. Harvested bunch should generally be collected in well padded
tray or basket and brought to the collection site. Bunches should be kept out of light after
harvest, since this hastens ripening and softening.
For local consumption, hands are often left on stalks and sold to retailers.
For export, hands are cut into units of 4-16 fingers, graded for both length and girth, and
carefully placed in polylined boxes to hold different weight depending on export
requirements.
Post harvest operations
At collection site injured and over mature fruits are discarded and for local market bunches
should be delivered through lorries or wagons. However, for more sophisticated and export
market where the quality is predominant, bunches should be dehanded, fruits are cleared in
running water or dilute sodium hypochlorite solution to remove the latex and treated with
thiobendasole; air dried and graded on the basis of size of fingers as already stated, packed in
ventilated CFB boxes of 14.5 kg capacity or as per requirement with polythene lining and
pre-cooled at 13-15ºC temperature and at 80-90% RH.
Such material should than be sent under cool chain at 13ºC for marketing
Yield
The planted crop gets ready for harvest within 11-12 months of planting. First ratoon crop
would be ready by 8-10 month from the harvesting of the main crop and second ratoon by 8-9
months after the second crop.
Thus over a period to 28-30 months, it is possible to harvest three crops i.e. one main crop
and two ratoon crops. Under drip irrigation combined with Fertigation yield of Banana as
high as 100 T/ha can be obtained with the help of tissue culture technique, even similar yield
in the ratoon crops can be achieved if the crop is managed well.
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 retain this
potential also after storage1. 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. In the recent past, however, other micropropagules like shoot buds, shoot tips,
organogenic orembryogenic 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 organogenesisand 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. Somatic embryogenesis, organogenesis and
enhanced axillary bud proliferation systems are the efficient techniques for rapid and
largescale 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 material as they are potent structures for plant regeneration either after having minor
treatment or without any treatment with growth regulator(s). Because the naked
micropropagules are sensitive to desiccation and/or pathogens when exposed to natural
environment, it is envisaged that for largescale 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 the in vitro derived propagules into ‘synthetic seeds’ or ‘synseeds’
or ‘artificial seeds’ The encapsulation technology has been applied to produce 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. Production of artificial seeds has unravelled
new vistas in plant biotechnology. The synthetic seed technology is designed to combine the
advantages of clonal propagation with those of seed propagation and storage. Despite the fact
that the technology is an exciting and rapidly growing area of research in plant cell and tissue
culture, there are many limitations for its practical use. The purpose of this review is to
present a report on prospects and limitations of synthetic seed production. The subject has
been earlier reviewed in a different context by various researchers
The 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 protective coatings, that make
them inconvenient to store and handle. Furthermore, they are generally regarded tolack a
quiescent resting phase and to be 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 for production of synthetic
seeds. Later it was thought that the encapsulated synthetic seed should also contain growth
nutrients, plant growth promoting microorganisms (e.g. mycorrhizae), and/or other biological
components necessary for optimal embryo-to-plant development. A number of patents
covering the development of seed analogues have been issued. However, success of the
synthetic seed technology is constrained due to scarcity and undesirable qualities of somatic
embryos making it difficult for their development into plants. The choice of coating material
for making synseeds is also an important aspect for synseed production. Based on 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 polyoxyethylene glycol (Polyoxr) 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 petri dishes and leaving them on
the bench overnight to dry. Such types of synseeds are produced only in plant species whose
somatic embryos are desiccationtolerant. On the contrary, hydrated synthetic seeds are
produced in those plant species where the somatic embryos are recalcitrant and sensitive to
desiccation. Hydrated synthetic seeds are produced by encapsulating the somatic embryos in
hydrogel capsules. The production of synthetic seeds for the first time by Kitto and Janick8
involved encapsulation of carrot somatic embryos followed by their desiccation. Of the
various compounds tested for encapsulation of celery embryos, Kitto and Janick8–10 selected
polyoxyethylene which is readily soluble in water and dries to form a thin film, does not
support the growth of micro-organisms and is non-toxic to the embryo. Janick et al.3 have
reported that desiccated artificial seeds were produced by coating a mixture of carrot somatic
embryos and callus in polyoxyethylene glycol. The coating mixture was allowed to dry for
several hours on a Teflon surface in a sterile hood. The dried mixture was then placed on a
culture medium, allowed to rehydrate, and then scored for embryo survival. In 1984
Redenbaugh et al.11 developed a technique for hydrogel encapsulation of individual somatic
embryos of alfalfa. Since then encapsulation in hydrogel remains to be the most studied
method of artificial seed production. A number of substances like potassium alginate, sodium
alginate, carrageenan, agar, gelrite, sodium pectate, etc. have been tested as hydrogels
butsodium alginate gel is the most popular. Hydrated artificial seeds consist of somatic
embryos individually encapsulatein a hydrogel To produce hydrated synthetic seeds, the
somatic embryos are mixed with sodium alginate gel (0.5–5.0% w/v) and dropped
into a calcium salt solution [CaCl2 (30–100 mM), Ca(NO3)2 (30–100 mM)] where ion-
exchange reaction occurs and sodium ions are replaced by calcium ions forming calcium
alginate beads or capsules surrounding the somatic embryos. The size of the capsule is
controlled by varying the inner diameter of the pipette nozzle. Hardening of the calcium
alginate is modulated with the concentrations of sodium alginate and calcium chloride
as well as the duration of complexing. Usually 2% sodium alginate gel with a complexing
solution containing 100 mM Ca2+ is used and is found to be satisfactory5,7,12.
However, Molle et al.13 found that for the production of synthetic seeds of carrot, 1%
sodium alginate solution, 50 mM Ca2+ and 20–30 min time period were satisfactory for
proper hardening of calcium alginate capsules. They have suggested the use of a dual \nozzle
pipette in which the embryos flow through the inner pipette and the alginate solution through
the outer pipette. As a result, the embryos are positioned in the centre of the beads for better
protection. For the past several years other unipolar structures such as apical shoot tips and
axillary shoot buds as well as apolar protocorms or protocorm-like bodies and even
Undifferentiated embryogenic calli are also being employed in synthetic seed production The
technology of hydrogel encapsulation is also favoured for synthetic seed production from
these micropropagules. For production of synthetic seeds from apical shoot tips and axillary
shoot buds, these organs are usually first treated with auxins for root induction and then their
microcuttings (approximately 4 or 5 mm in length) areencapsulated in sodium alginate gel
following the method described by Redenbaugh et al. for alfalfa somatic embryos. However,
mulberry and banana plantlets were obtained from alginate-encapsulated shoot buds without
any specific root induction treatment. To avoid bacterial contamination Ganapathi et al.15
added an antibiotic mixture (0.25 mg/l) containing rifampicin (60 mg), cefatoxime (250 mg)
and tetracycline- HCl (25 mg) dissolved in 5 ml dimethyl sulphoxide to the gel matrix.
Activated charcoal (0.1%) was also added to the matrix to absorb the polyphenol exudates of
the encapsulated shoots of banana.
Banana Tissue culture and Synthetic Seeds
Progress in biotechnological research during the last two decades has opened up
unprecedented opportunities in many areas of basic and applied biological research.Plant
tissue culture, which is an important component of plant biotechnology, presents new
strategies for the improvement of cereals, legumes, forest trees, plantation crops and
ornamental plants. Besides, plant cell cultures provide a good system for many basic studies
in plant breeding, plant physiology, genetics and cell biology. Cell manipulations through the
sophisticated methods of genetic engineering for plant quality and product improvement has
to rely on plant tissue culture for the final goal. Micro propagation is an area of plant tissue
culture which has received maximum attention of researchers for its potential commercial
applications.
Plant tissue culture refers to growing and multiplication of cells, tissues and organs of plants
on defined solid or liquid media under aseptic and controlled environment. The commercial
technology is primarily based on micropropagation, in which rapid proliferation is achieved
from tiny stem cuttings, axillary buds, and to a limited extent from somatic embryos, cell
clumps in suspension cultures and bioreactors. The cultured cells and tissue can take several
pathways. The pathways that lead to the production of true-to-type plants in large numbers
are the preferred ones for commercial multiplication. The process of micropropagation is
usually divided into several stages i.e., prepropagation, initiation of explants, subculture of
explants for proliferation, shooting and rooting, and hardening. These stages are universally
applicable in large-scale multiplication of plants. The delivery of hardened small
micropropagated plants to growers and market also requires extra care.
The regeneration of plants through the techniques of plant tissue culture and their subsequent
acclimatization and delivery to the field poses many problems to make tissue culture
technology a viable alternative proposition. The successful demonstration of encapsulation of
tissue culture derived propagules in a nutrient gel has initiated a new line of research on
synthetic seeds. Synthetic seeds are basically defined as, "encapsulated somatic embryos
which functionally mimic seeds and can develop into seedlings under sterile conditions". In a
broader sense, it would also refer to encapsulated buds or any other form of meristems which
can develop into plants.
Many plant systems are known to produce abundant number of embryos in culture which
share many properties similar to natural embryos including germination leading to plant
production. To mimic the
natural seeds, embryos from cultures are encapsulated in a nutrient gel containing essential
organic/inorganic salts, carbon source, plant hormones and antimicrobial agents and coated
completely to protect the embryos from mechanical damages during handling and to allow
the development and germination to occur without any undesirable variations. Several agents
have
been attempted for encapsulation and sodium alginate complexing with calcium chloride is
found to be the most suitable. Bythis method, two types of synthetic seedsare prepared:
hydrated and desiccated.Hydrated synthetic seeds consist ofembryos individually
encapsulated in ahydrogel, whereas in desiccated type thecoating mixture is allowed to dry
for severalhours in a sterile hood.The Plant Cell Culture Technology Group ofNuclear
Agriculture and BiotechnologyDivision had initiated research on syntheticseeds in the late
1980s working withsandalwood and mulberry. Eventually other crop systems such as banana,
cardamomand rice have also been taken up for theproduction of synthetic seeds.
I (1-3) : Synthetic seeds and plantlets in mulberry and banana. 1- synthetic seeds ofmulberry planted in soil; 2- mulberry synthetic seeds germinating into plantlets in soil; 3-completeplantlets of banana obtained from synthetic seeds (arrow indicates portion of the synthetic seedstill attached to the plantlet)
Banana is an economically profitable cropwith a large consumption in the country and a
considerable export potential. Ediblebananas are vegetatively propagated by suckers as viable
seeds are generally notproduced in these cultivars. New and effective means of propagating
banana would be advantageous over conventional use of sucker material for germplasm
maintenance, exchange and transportation. Shoot tips excised from the aseptically raised
shoot cultures were excised and encapsulated to prepare synthetic seeds. High percent
germination of these synthetic seeds was achieved on a very simple nutrient medium.
Addition of the extract of blue green algae to the nutrient matrix enhanced germination
frequency. A cell mass (callus) initiated from male flower buds produced embryos which
have been successfully encapsulated and germinated. Hence, a twin facility is available in
banana to either encapsulate shoot apices or embryos.
The examples presented above suggest that, by employing synthetic seeds, the tissue culture
raised plants can be regenerated on a simplified medium eliminating subcultures, thus
reducing the cost of operation. Development of protocols for direct recovery of plants from
synthetic seeds under non sterile conditions may havea greater impact. Although large
number of plants can be produced in tissue cultures through embryogenesis / multiple shoot
cultures, their delivery is cumbersome.
Embryos or shoots have to be separated singly and transferred for rooting to achieve root
shoot balance, and the plants have to be hardened in the green house before field planting.
Direct sowing of synthetic seeds in the soil does not need acclimatization often required for
the tissue cultured plants. It thus provides an ideal delivery system enabling easy flexibility in
handling and transport as compared to large parcels of seedlings or plants. For large scale
commercialization in synthetic seeds technology, enhanced production of propagules is
necessary. Current tissue culture methods do not generate adequate propagules and are not
sufficient to meet the demands of commercial exploitation of synthetic seeds technology.
Standardization of methods for synchronization of developing propagule followed by
automation of the whole process of sorting, harvesting, encapsulation and germination of the
coated propagules can enhance the pace in the production of synthetic seeds.
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