1. Somatic Embryogenesis
LECTURE 5
Somatic Embryogenesis
Dr. Aparna Islam

2. Embryogenesis: Zygotic
Embryogenesis: The process of embryo initiation and development.
Zygotic embryogenesis: Initiation and development of embryo as a result of fertilization.
Embryo: A very young plant developing inside the female gametophyte.

4. Embryogenesis: Somatic
Somatic embryogenesis: The process of a single cell or a group of cells initiating the developmental pathway that leads to reproducible regeneration of non-zygotic embryos capable of germinating to form complete plants
Somatic embryogenesis: Regeneration of embryo from somatic (vegetative) or non-sexual part of the body.
Under natural conditions this pathway is not normally followed, but from tissue cultures somatic embryogenesis occurs most frequently and as an alternative to organogenesis for regeneration of whole plants.

6. Kohlenbach (1978) proposed the following classifications of embryos:
Embryos formed in cultures have been variously designated as accessory embryos, adventive embryos, embryoids and supernumerary embryos.
Kohlenbach (1978) proposed the following classifications of embryos:
1. Zygotic embryo: those formed by fertilized egg or the zygote.
2. Non-zygotic embryo: those formed by sporophytic cells (except zygote) either in vitro or in vivo. Such somatic embryos arising directly from other embryos or organs (stem embryo in carrot) are termed adventive embryos.
Embryoid: Non-zygotic embryo formed in the culture. It is also called Embryo.
Parthenogenetic embryos: those formed by unfertilized egg.
Androgenic embryos: those formed by the male gametophyte (microspore or pollen grain).

7. What is the Differences Between Zygotic and Somatic Embryo?
Requirement of fertilization
Characters variability with the parent plant
Originating from soma cell or reproductive cell
Developed from pre-embryonic determined cells (PEDC) or from induced embryonic determined cells (IEDC)
Stages of embryogenesis or early divisions of embryo development
Seed formation
Dormant phase before germination
Secondary embryogenesis

8. Process of Somatic Embryogenesis
Two Types : Direct and indirect somatic embryogenesis.
Explants
DIRECT S.E.
INDIRECT S.E.
Placed on Solid medium supplemented with 2,4-D for callus production
Placed in medium (liquid or solid) supplement with 2,4-D
Callus transferred to liquid medium with high 2,4-D
After initiation of embryos old medium replaced by less or no 2,4-D supplemented medium for maturation of the embryo

9. Somatic embryogenesis is initiated either by ‘pre-embryonic determined cells’ (PEDCs) or by ‘induced embryonic determined cells’ (IEDCs).
In PEDCs, the embryonic pathway is predetermined and the cells appear to only wait for the synthesis of an inducer or removal of an inhibitor to resume independent mitotic divisions in order to express their potential.
Such cells are found in embryonic tissues, tissues of young in vitro grown plantlets, nucellus and the embryo sac.

10. In case of IEDCs, it requires re-determination to the embryogenic state by exposure to specific growth regulators, such as 2,4-D. These cells are differenentiated generally in microspore (anther) cultures and callus cultures. Once the embryogenic state has been reached both cell types proliferate in a similar manner as embryogenic determined cells (EDCs).

11. Steps of Somatic Embryogenesis
Induction
2. Development
3. Maturation
4. Germination

12. Explants: Totipotent Embryonic cells collected from embryonic or young seedling tissue and young inflorescence (before maturation on the floral primordia), young shoot tip, young root, immature leaf.
Medium: can be MS or any suitable basal medium of solid or liquid consistency. Usually the first culture is made in the solid medium while the subculture of embryonic callus is done with liquid medium.
The in vitro development of somatic embryo was first observed in carrot suspension cultures in 1958 by Steward and associates, although Ruinert (1958, 1959) also induced somatic embryogenesis in callus cultured in semi-solid medium.

13. Induction At this stage ‘Embryogenic Clumps’ are form
Hormonal requirement
Auxins required for induction
presence of auxin is generally essential for embryo initiation.
Eg. 2,4-D (most used), NAA and IAA (some time)
At this stage ‘Embryogenic Clumps’ are form
Embryonic clumps : this is a stage before the embryo development. At this stage the cell divides repetitively that leads to production of groups of cells that develop into small clusters of meristematic cells from which embryos will arise.
Complex Nutrient Supplements:
Variation in requirement occur species by species manner. Basal media with malt extract (500mg/l) gives somatic embryo in Citrus nucellar cells.

14. Induction (cont.)
Sometimes, other nutrient supplements are also needed for induction.
Nitrogen source:
A substantial amount of nitrogen, usually in reduced form such as ammonium salts, is needed for both embryo initiation and maturation. In MS medium high levels of nitrogen is present in the form of ammonium nitrate. Variation to this is also present. Eg. In carrot, somatic embryos are formed when high concentrations of inorganic nitrogen (nitrate) is supplied to the culture medium.
Other constituent:
Some time other nutrient may be important for embryogenesis. Eg. for wild carrot high concentration of potassium, presence of activated charcoal are important.

15. Induction (cont.) Culture methodology
To obtain significant amount of callus to have sufficient amount of somatic embryo, the callus induced in the induction stage needs to be proliferated. Usually, the culture is placed in liquid medium at this point for establishment of Embryonic Suspension Culture.
To initiate suspension culture, 2.5 to 3.0 grams callus tissue is placed in 50 ml of the growth medium and the flasks are agitated on a shaker platform at rpm. Embryonic cell suspension developed within 3-4 weeks needs for sub-culture in fresh medium at periodic intervals.
Frequent sub-cultures not only prevent cells from rapid senescence at the end of the growth phase, but maintain embryonic potential by minimizing the extent of chromosomal changes in the cell cultures.

16. Induction and further
Sub-culture: Suspensions are subcultured every 4 weeks by transferring 5 ml of suspension to 65 ml of fresh medium (1:13).
Somatic embryogenesis is induced by cultivating either callus pieces or portions of suspension in the same medium without 2,4-D.
Within 3-4 weeks numerous embryos in different stages of development appear in culture flasks.

17. Induction (cont.). Somatic embryogenesis is achieved in two steps:
First: the callus is initiated and multiply on auxin rich medium (2.4-D mg/l), which induced the differentiation of localized groups of meristematic cells called embryonic clumps (ECs). This medium is called PROLIFERATION MEDIUM (PM).
Second: ECs develop into mature embryos when transferred to very low ( mg/l) or no auxin medium for the maturation. This medium is called EMBRYO DEVELOPMENT MEDIUM (EDM).

19. Development Auxin must be removed for embryo development
Continued use of auxin inhibits embryogenesis
Stages of somatic embryo development:
Globular
Heart
Torpedo stage

20. Development
Various stages of somatic embryos are as follows
Globular stage: Large group of cells not yet having a definite embryoid shape.
Heart shape: A characteristic three lobbed stage where cotyledonary initials are separated from the root pole.
Torpedo shape: an elongated form of heart shaped embryo which resemblances the plantlet that will be formed in the next (final) stage.

21. Development Globular Heart Torpedo Maturation
Soybean – Wayne Parrot, UGA

22. Maturation
Require complete maturation with apical meristem, radicle, and cotyledons
Often obtain repetitive embryony
Storage protein production necessary
Often require ABA for complete maturation

23. Germination May only obtain 3-5% germination
Carbon sources: Sucrose (10%), mannitol (4%) may be required

24. Maturation and Germination (Conversion)

25. Application of Somatic Embryogenesis
1. Clonal propagation
Both the growth of embryonic cells and subsequent development of somatic embryos can be carried out to have continuously propagules which are true-to-type of parent. Here the system is adopted where repetitive somatic embryogenesis cycle is initiated from previously existing somatic embryos in order to produce clones. Moreover, nucellus embryos are free of virus and can be used for raising virus free clones.

26. 2. Raising somaclonal variation
Embryo formed directly from PEDCs produce relatively uniform clonal material.
However, embryos formed by the indirect method involving IEDCs callus generates a high frequency of somaclonal variants. Thus may regenerate a new strain of plant.
Mutation during adventives embryogenesis may give rise to a mutant embryo which on germination would form a new strain of plant.
Somaclonal Variation: Genetic variations in plants that have been produced by plant tissue culture and can be detected as genetic or phenotypic traits.

27. 3. Synthesis of artificial seeds
Somatic embryo enclosed in a protective coating,
Advantages: ‘low-cost-high-volume’ propagation system; production of many seeds at a time; and the use of conventional seed handling techniques for embryo delivery. The objective is to produce clonal ‘seeds’ at a cost comparable to true seeds.
Two types of artificial seeds have been developed,
Hydrated seeds: mixing embryos with sodium alginate, followed by dropping into a sol. of calcium chloride to form calcium alginate beads.
Eg. Alfalfa, cauliflowers. About 29-55% embryos encapsulated with this hydragel germinated and formal seedlings in vitro.

28. Desiccated seeds: mixing of equal volume of clump of embryos in suspension and 5% solution of polyethylene oxide (a water soluble resin), which dry and form poly-embryonic desiccated wafers.
The survival of encapsulated embryos can be further increased by embryo ‘hardening’ treatment with 12% sucrose, followed by chilling at high inoculation density.

29. Why Encapsulation is essential .?
Alginate protects Synthetic seeds from microbial infection.
Protect the seeds from the mechanical damages during handling.

30. Advantages of Somatic Embryogenesis
Physiological study
Artificial seed production
Transformation of economical important plants species eg. peanut, cotton
Plantlet production in large number for commercialization
Embryogenesis can be continuous for and give rise to secondary or more times

31. Disadvantage of Somatic Embryogenesis
In this process there is a high possibility of rising mutation
The method is difficult
The repeated subculture the chance of loosing regenerative capacity become greater

32. 4. Genetic transformation
Transformation of somatic embryos and raising plantlets from those following repetitive embryogenesis may raise to numerous transgenic plant lines.

33. In vitro Development of Cauliflower Synthetic Seeds and Development of Plantlets In vivo
Zahid Qamar, Md. Belal Hossain, Idrees A. Nasir, Bushra Tabassum and Tayyab Husnain
Plant Tissue Cult. & Biotech. 24(1): 27‐36, 2014 (June)

34. Introduction
Cauliflower (Brassica oleracea var. botrytis) is one of the most cultivated vegetables in the world
Recently found to be useful in the prevention of cancer
Problems in the production of a uniform and quality crop of cauliflower

35. Why Cauliflower? Open pollinated crop
There are technical challenges to producing a reliable self incompatible inbred lines.
Major problems in cauliflowr cultivation is the quality of seed production
Strong demand for improved seed biotechnology resulting in efficient and stable regeneration method

36. Objectives
Establish an efficient, inexpensive callus induction protocol from hypocotyls for cell suspension to produce cauliflower synthetic seeds
With a prolonged period of viability(assessment of germination viability of synthetic seeds)
Cable of germinating
Developing into viable plants

37. The steps of the experiment
Explants Isolation
Callus Induction
Establishment of cell suspension
Synchronization
Encapsulation
Embryo Hardening
Shoot Regeneration
Germination Viability
Plantlet Development
Hardening

38. Explant Isolation
Explant used here is the hypocotyl of the cauliflower seed

39. Explant Isolation Seed Sterilization 0.1% HgCl2 5mins
Wash with distilled water 3 times 5mins each
Plating for Germination
25 ± 2°C dark incubation
Explant (Hypocotyl) cm long pieces from 5-7 days old seedlings

40. Callus Induction Hypocotyl explant (from 5-7 days old seedlings)
Cut into cm pieces
Placed horizontally into MS media
with hormones+ sucrose + phytagel to solidify
pH adjusted to before autoclave
Sub-cultured at 7 days interval (first 3-5 days in dark, then 16 hrs light and 8 hrs dark period)

41. Results After 15 days embryogenic calli found
Yellowish, friable callus with globular, dense cytoplasmic cells
Right conc. and ratio of BAP and 2,4-D (2mg/l 2,4-D mg/l BAP )
Sucrose: – 20, 30, 40 g/l of media tested
– 30 g sucrose best for callus induction

42. Suspension Culture Callus suspended in callus medium for 7 days
Sub-cultured in same medium without 2,4-D (it was found that continued use of auxin inhibits embryogenesis)
mg calli in 30 ml cell suspension media. Rotary shaker (90 rpm), 16 hrs light, 22 +/- 2 °C
Cells regularly observed under microscope

43. Heterogenous cell suspension
2 types of cells clearly observed under microscope
1. Elongated, vacuolated, larger cells without starch
(10-15 %)
2. Globular, round, smaller cells, with dense cytoplasm, nucleolus like body, rich in starch
(85-90 %)
Fig: Microscopic views of the suspended cells in suspension culture

44. Cell type 1 was discarded by sieving through 150 µm
If sieved at intervals longer than 7 days, number of cell type 1 increase
Suspended cells went through different stages of embryogenesis
Globular cells → heart → torpedo → cotyledonary shape
Fig: Globular cells turned into heart or angular shaped

45. Shoot regeneration
To check the shoot regeneration response of established embryogenic calli
Calli were placed in various shoot regeneration media
MS nutrients:
30 μM adenine sulphate
3 μM thiamine HCl
580 μM NaH2PO4
And containing combination of Plant Growth Hormones:
4 ‐ 6 mg/l BAP
2 ‐ 4 mg/l Kn and
4 ‐ 6 mg/l GA3

46. Optimal Regeneration Results :
2 mg/l Kn
6 mg/l GA3
5 mg/l BAP
MS vitamin
Figs. Shoot regeneration from embryogenic callus (1, 1a) and cell suspension cultures

47. Synchronization
Suspended cells synchronized by Giuliano et al Method (1983).
After 2 to 3 times successive sub culturing, At weekly interval 84% cell found at Globular stage.
Cells are transferred to the modified suspension media containing BAP only.
2-3 days microscopic studies- 71% cells turned into heart and torpedo shaped embryos.
Subsequent sub culturing on MS supplement with BAP and NAA 28% embryos become mature.
Globular Stage
Torpedo Stage
Somatic Embryos

48. Synchronization Stage

49. Encapsulation Sodium Alginate is used for the mature embryos coating .
(Sodium Alginate +Mature Cell Suspension ) ratio 1:4 dropped into 100mM Calcium Chloride Solution for getting synthetic seeds.
Synthetic seeds – artificial encapsulation of any cells that have the capability to form a plant in in vitro or ex vitro.
- Can be stored for a long period.
-Easy to carry for long distance.

50. For Hardening the synthetic seeds-
Rinsed in Double distilled Water.
Immersed in MS- Containing 3% sucrose, MS Fortified with 5 mg/l BAP, 2 mg/l Kn, 6 mg/l GA3 and MS Vitamin.
Incubated at Gyratory shaker for min.
Mature embryos Turned into Complete seeds.
Sodium Alginate complex with Calcium Chloride solution used –
- Viscosity.
- Low Toxicity.
- Quick Gelatinization.

51. Germination Synthetic seeds were stored at 40C
For germination assessment, neutral gel consisting phytagel dissolved in autoclaved tap water was used
Germination viability was studied

52. Germination Viability Assessment
Fig. Germination viability percentage of cauliflower synthetic seeds developed by encapsulating mature somatic embryos by sodium alginate.
Successful germination of 83.5% cauliflower synthetic seeds
Germination efficiency decreased to 50% after 12 weeks of storage
By the 16th week, the rate of germination dropped to 0%

53. Short comings: Prolonged period of viability not established
Germination efficiency decreased to 50% after 12 weeks of storage
By the 16th week, the rate of germination dropped to 0%

54. Plantlet development
Germinated synthetic seeds were transferred from the Petri plate to test tube containing 3 different shoot and root regeneration media
Fig 7. Cauliflower synthetic seeds germinated on neutral gel media.
Fig 8. Cauliflower plantlets with regenerated shoots and roots derived from synthetic seeds.
Fig 9. Complete cauliflower plantlets derived from synthetic seeds.

55. Acclimation
Complete plantlets regenerated from synthetic seeds were transferred into pots
Containing autoclaved compost soil prepared by thoroughly mixing clay, sand and compost in the ratio of 1 : 1 : 1 respectively.
The plants were completely covered with plastic bag for 2 weeks (to maintain the humidity)
Then progressively exposed to normal environmental conditions for acclimation.

56. Agrobacterium-mediated transformation and transgenic cotton (Gossypium hirsutum) production through embryogenesis
Regeneration protocol:
Author:R Kumaria et al.
Issue date: 12/02/2003
Agrobacterium-mediated
transformation & transgenic
plant production
Author: S. Leelavathi et al
Issue date: 17/09/2003
Journal: Plant Cell Rep
Issue: 21:
Issue: 22:

57. Probems associated with previously established somatic embryogenesis protocols
prolonged culture period
high frequency of abnormal embryo development
low conversion rate of somatic embryos
lack of shoot elongation.

58. Regeneration protocol
Cotton variety: Coker 310
Explants: Hypocotyl and
Cotyledonary leaf
Explant preparation
Callus induction
Isolation of embryogenic callus
Induction of somatic embryos
Germination and maturation of embryos
Development of plantlets
Establishment of plants in the soil

59. Obtaining embryogenic calli
Callus obtained when explant cultured on MS medium with 0.1 mg/l 2,4–D, 0.5mg/l Kn and 3% maltose (8 weeks)
Embryogenic callus formed when callus sub-cultured on MS medium with 3% maltose
Frequency of embryogenesis improved on hormone free MS medium with higher amount of potassium nitrate.
Globular-stage embryos were produced on this medium.

60. Acceleration of differentiation of globular stage embryos into plantlets through manipulation of METABOOLIC STRESS (nutrient and microenvironment conditions).
Stresses :
The embryogenic calli were plated directly onto media (full strength, ½ strength and 1/5 strength MS media)
Or on sterile filter paper placed on the various MS media concentrations
Culture plates sealed with Parafilm (to keep high humidity in culture plates) or with porous sealing tape that causes rapid drying of medium (low humidity)

61. Results of stress on development and maturation of embryos
The results suggest that dilution of medium helps differentiation of embryonic calli into globular stage.
However, for further maturation to torpedo stage and cotyledonary stage embryos higher nutrient concentration is needed.
For germination of cotyledonary embryos and maturation to plant, higher nutrient level is a prerequisite.
Both filter paper and porous tape improved development and maturation

62. Somatic embryogenesis and plant regeneration in cotton
Fig a: Embryogenic calli on High nitrate medium (x4).MS + 1,900 mg/l KNO3
Fig b: low-frequency differentiation of globular embryos on basal medium. (x3.5).
Fig c: abnormal embryos from long term cultures (x0.9)
Fig d: initiation of large no. of globular embryos on basal medium supplemented with 1% maltose (x5.5)
Fig e: globular embryos developed on 1/5 strength MS medium (x4.9)
Fig f: somatic embryos at various stages of development (x1.4)
Fig g: cotyledonary embryos (x3)
Fig h: germination of embryos (x1.1)
Fig i: regenerated plants (0.7)

63. Why do we need to improve cotton (Gossypium hirsutum)?
Highly susceptible to abiotic stress, viral and fungal infections and Insect predation
Hence need to improve productivity for insect pest management, as well as fiber quality
Why do we need to improve cotton (Gossypium hirsutum)?

64. Agrobacterium-mediated transformation protocol for cotton
Transformation of insecticidal protein coded by cry1Ia5 gene.
Cotton variety: Coker 310
This toxin gives complete protection against predation by Heliothis armigera in tobacco.
Agrobacterium tumefaciens strain LBA4404 used, cry1Ia5 gene on binary construct under CaMV 35S promoter and nopaline synthyetase gene terminator.
Explant: Emvbryogenic calli

65. Comparison of protocols between previously established and presently used

66. Agrobacterium–mediated Transformation Experiment
Embryogenic calli were infected with the bacteria with intermittent vigorous shaking for 20 minutes and bacterial suspension removed
Co-cultured in dark on basal MS medium for 36 hours
Transferred to filter paper on selection medium and plate sealed with porous tape. Cultured for 3-5 weeks
Regeneration: Growing clusters seperated as each represented separate transformation event
Regeneration protocol

67. Result of Agrobacterium–mediated Transformation Experiment
Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene and cultured under dehydration stress and antibiotic selection for 3-6 weeks
75 clusters observed on selection plates, these embryos were cultured on multiplication media and then cotyledonary embryos developed on embryo maturation medium to obtain 12 plants per plate on average. 83% of these confirmed to be transgenic by southern blot anaylsis. An efficiency of 10 kanamycin resistant plants per petri dish of co-cultivated emrbyogenic callus was seen.

68. Transformation of embryogenic calli of cotton and subsequent regeneration
a: Embryogenic cotton calli used for transformation. b: Enlarged view of embryogenic calli used for transformation c: Co-cultivated cotton calli on selection medium after 6 weeks showing a large number of globular embryo clusters d: Enlarged view of one of the globular embryos from c. e. Kanamycin resistant transformed cotyledonary embryos on embryo maturation medium f. Kanamycin-resistant plants in soil pots in green house.

69. Concluding remarks
Embryogenic callus provide large population of embryogenic competant cells that are extremely amenable for transformation.
Each transformed cell represents an independent transgenic line, tremendously increasing the number of transformation events in a regenerable tissue.
Protocol is economic, rapid, less laborious and has high efficiency of transformation