this ppt give idea about somatic emryogenisis and syntetic seed production

1. Embryogenesis for hybrid production
Saurav Saha
2014-11-106

2. Introduction
• Embryogenesis is a process where a plant or embryo is
derived from a single somatic cell or group of somatic
cells
• Direct embryogenesis occurs when embryos are
started directly from explants tissue creating an
identical clone
• Indirect embryogenesis occurs when explants
produced undifferentiated, or partially differentiated
callus

4. Practical Application of Somatic
Embryogenesis
• Clonal Propagation
• Raising Somaclonal Variants in Tree Species
• Synthesis of Artificial Seeds
• Source of Regenerable Protoplast System
• Genetic Transformation
• Synthetic of Metabolites
• Used in hybrid production

5. Overcome the interspecific barrier
• Arachis hypogaea × A. stenosperma
are in viable due to pre and post zygotic
barrier
 It is overcome by the somatic embryogenesis
process
 Immature embryo are rescued and
germinated in-vitro

6. Embryo culture process
• When embryo are heart shape stage
• Normally 7-12 days after pollination
• Dip the pod in a 70% alchohal for 1 min and then
starilized with Naocl 20% for 10 min wash by dw
• Embryo are carefully collected from the immature
seed by the forceps
• Inoculate in following media MS+ casein
hydrolyses(200mg/l)+ L-Glumatic ( 100-200mg/l)
+NAA( 0.01 micromolar) + BAP( 1.0 micromolar)+
sucrose ( 3- 8%)

7. Conti…..
• Incubate at 25°C
• Transfer the plantlet after 3-4 week in half MS media
without growth regulator with reduced 1% sucrose
• Transfer the plant for hardening unit after 2-3 weak
of subculture

8. Production of interspecific somatic hybrid
Asparagus
• Protoplasts of A. officinaiis and A. macowanii
were both prepared from calli
• 2% (w/v) Cellulose 0.5% (w/v) Maceroxyme R-10
0.05% (w/v) Pectolyase Y-23 10 mM CaC12.2H,0, 5
mM MES and 0.6 M sorbitol, at pH 5.7
• Protoplasts of both species were mixed at a ratio
of 1: 1
• Electrofusion was carried out using an Electra Cell
Manipulator 401 A

9. • 0.1% (w/v) gellan gum-solidified l/2 MS medium
containing 1 mg/I naphthaleneacetic acid (NM), 0.5
mg/l zeatin, 1 g/l glutamine and 0.6 M glucose are
use for protoplast culture
• After 2 months of culture, colonies weretransferred to
MS medium containing 2 mg/l2,4- D, 3% sucrose and
0.2% (w/v) gellan gum,
• cultured to the liquid MS medium without plant
growth regulators for removal of auxin for somatic
embryogenesis

10. Conti…
• Treated for 14 days at 4°C in the dark on 1.0% (w/v)
gellan gum-solidified MS media
• MS medium consisting of half strength-MS salts,
vitamins, 3% (w/v) sucrose and 0.2% (w/v) gellan
gum are use as germination media

11. Production of Brassica hybrid
• Brassica F1 hybrid produced by cross between
turnip rape and R500
• Bud are collected from the F1 hybrid and surface
sterilize it
• The basic media supplement with 100mg/l serine
• Culture condition should be 25°C continuous and
then 35°C for3, 7, or 14 days prior to transfer to
25°C

12. Conti…
• Culture was maintained in darkroom and
observation should taken after one weak of
culture
• Count the number of anther produce embryo
• Embryo are removed from the anther
• Culture at 25°C in hormone free B5 media
contain 2% sucrose
• Plant developed directly from the media and
maintain in a mist chamber for the 7-4 days
before hardening

14. Some example of embryo rescue for somatic
embryogenesis
• For the first time successful embryo culture to
obtain an interspecific cross between Linum
perenne × L. austriacum was demonstrated by
Laibach
• Interspecific hybridization of red clover (Trifolium
pratense L.) with T. sarosiense Hazsl. Using in
vitro embryo rescue
• Somatic embryogenesis and plant development
from immature zygotic embryos of seedless grapes
(Vitis vinifera L.)

15. FACTORS AFFECTING SOMATIC
EMBRYOGENESIS
• Immature zygotic embryos( in cereals)
• Genotype
• Growth regulators
- 2 4 D 0.5-1 mg / l for callusing
- if embryo need concentration will be low(0.01-0.1 mg/l
o PH should be reached to 5.6

16. Synthetic seed
• Naked embryo may be use for as a planting
material by making a protective cover around
the seed called synthetic seed
Kitto and Janick (1982) 1st developed the synthetic sees of
carrot
• Currently two types of synthetic seeds are being
developed
(1) Desiccated
(2) Hydrated.

17. Desiccated synthetic seeds
• polyoxyethylene (Polyox r) used a chemical for
encapsulation
• Readily soluble in water and dries to form a
thin film,
• Does not support growth of microorganism
and is non-toxic to the embryos

18. Conti…..
o SEs with polyox equal volumes of embryo
suspension and a 5% (w/v) solution of polyox
• were mixed to give a final concentration of 2.5%
polyox
• The suspension was dispensed as 0.2 ml drops
from a pipette on to Teflon sheets and dried to
wafers in a laminar flow hood.
• This type most commonly use in carrot seed
production

19. Hydrated synthetic seeds
• In 1984 Redenbaugh et al. developed a technique for
encapsulation of single, hydrated SEs of alfalfa.
• SEs are mixed with 2% (w/v) solution of Na-alginate
and dropped
• Dropped, using a plastic pipette, into a 100 mM
solution of Ca(NO3)2.
• ion exchange reaction occurs and sodium ions are
replaced by calcium ions forming Ca-alginate.