Embryogenesis for hybrid production

Saurav Saha

2014-11-106

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

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

Overcome the interspecific barrier

• Arachis hypogaea × A. stenosperma

are in viable due to pre and post zygoticbarrier

It is overcome by the somatic embryogenesisprocess

Immature embryo are rescued andgerminated in-vitro

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%)

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

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

• 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

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

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

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

Some example of embryo rescue for somatic

embryogenesis

• For the first time successful embryo culture to obtain an interspecific cross between Linumperenne × 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.)

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

Synthetic seed• Naked embryo may be use for as a planting

material by making a protective cover aroundthe 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.

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

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

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 mMsolution of Ca(NO3)2.

• ion exchange reaction occurs and sodium ions are replaced by calcium ions forming Ca-alginate.