Work funded by the Spanish national project AGL2010and the Madrid Regional R&D program S2009AMB

Acknowledgements: Araceli Hernández and Celina Villarreal for their technical assistance

Cristina Celestino, Mª Mar Ruíz, Nieves Alonso, Jesús Alegre, Mariano Toribio

Cloning Stone pine (by somatic embryogenesis

Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario (IMIDRA)Finca “El Encín”. Apdo. Postal 127, 28800 Alcalá de Henares (Madrid)

Work funded by the Spanish national project AGL2010-22292-CO03-01,and the Madrid Regional R&D program S2009AMB-1668 (REGENFOR-CM)

Acknowledgements: Araceli Hernández and Celina Villarreal for their technical assistance

Cristina Celestino, Mª Mar Ruíz, Nieves Alonso, Jesús Alegre, Mariano Toribio

Cloning Stone pine (Pinus pinea L.)by somatic embryogenesis

Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario (IMIDRA)Finca “El Encín”. Apdo. Postal 127, 28800 Alcalá de Henares (Madrid)

EL ENCÍNAlcalá de Henares

Celestino et al. Cloning Stone pine (Pinus pinea

OUTLINE

- Vegetative propagation and somatic embryogenesis

- Multivarietal Forestry (MVF)

- IUFRO Unit 2.09.02: Somatic embryogenesis and Vegetative propagation technologies

- Our work on the Stone pine

Pinus pinea L.) by somatic embryogenesis

OUTLINE

Vegetative propagation and somatic embryogenesis

Multivarietal Forestry (MVF)

IUFRO Unit 2.09.02: Somatic embryogenesis and Vegetative propagation technologies

Our work on the Stone pine

Populus euphraticaShoot sprouts from roots

“Pando” (Populus tremuloides) more than 40.000 trees from the same root system

Natural vegetative propagation

Populus euphratica: Shoot sprouts from roots

) more than 40.000 trees from the same

Natural vegetative propagation

Fruit trees:CitrusMalusPrunusetc.

GrapevinesVitis

Fruit trees:CitrusMalusPrunusetc.

Olive treesOlea

VEGETATIVE PROPAGATION OF SEVERAL WOODY CROPS

Clonal plantations of Clonal plantations of Eucalyptus in Brazil

The development of biotechnologiesas somatic embryogenesisvarietal forestry (MVF)regeneration by SE, combinedoffers an opportunity to developvarieties.

Multi-varietal ForestryThe use of tested tree varieties in plantation forestry,

balancing genetic gain and variability

Clone = the ortet of any genotypeVariety = a clone selected and tested for certain attributes

biotechnologies of propagation such(SE) enables to put multi-

(MVF) into practice. Plantcombined with cryopreservation,

develop highly valuable clonal

varietal Forestry (MVF)

tree varieties in plantation forestry, balancing genetic gain and variability

Clone = the ortet of any genotype and its copies or rametsVariety = a clone selected and tested for certain attributes

SOMATIC EMBRYOGENESIS IN CONIFERS

Picea abies(Chalupa, 1985; Hackman and von Arnold, 1985)

Larix decidua(Nagmani and Bonga, 1985)

The SE process in conifers involvescontinuous growth of embryo-immature zygotic embryos, differentiationand germination to give somatic seedlings

Pinus taeda: photos courtesy of

involves the initial outgrowth and-suspensor masses (ESM) from

differentiation into somatic embryos,seedlings.

: photos courtesy of Mark Rutter (ArborGen)

Silvagen Inc.BC Research and Innovation ComplexSilvagen Inc.BC Research and Innovation Complex

Commercial Production

CellFor, Inc., Victoria, Canada

• High-value SE-derived varieties of loblolly pine– 6 million in 2006– 24 million in 2007– Sold in the Southern U.S.

ArborGen, Summerville, USA

Commercial Production

Victoria, Canada

derived varieties of loblolly pine

Park, 2007

Romero JL – CELLFOR (2007) Producción comercial de embriogénesis somática. Biotecnologia Florestal, Biowork IX, Brasil

CELLFOR (2007) Producción comercial de Pinus taeda usando embriogénesis somática. Biotecnologia Florestal, Biowork IX, Brasil

Romero JL – CELLFOR (2007) Producción comercial de embriogénesis somática. Biotecnologia Florestal,

CELLFOR (2007) Producción comercial de Pinus taeda usando embriogénesis somática. Biotecnologia Florestal, Biowork IX, Brasil

Romero JL – CELLFOR (2007) Producción comercial de embriogénesis somática. Biotecnologia Florestal, Biowork IX, Brasil

CELLFOR (2007) Producción comercial de Pinus taeda usando embriogénesis somática. Biotecnologia Florestal, Biowork IX, Brasil

Our work on the Stone pine

Stone pine clonal bankNational Forest Breeding Centre “Puerta de Hierro”,

Madrid. Spanish Ministry of Environment.

Our work on the Stone pine

Stone pine clonal bankNational Forest Breeding Centre “Puerta de Hierro”,

Madrid. Spanish Ministry of Environment.

Initiation and proliferation of ESM:

G Proliferation as

Estimation of growth by fresh weight increasing. A initial 100 mg of fresh ESM after two weeks resulted in average:

G 3-fold increase in FW as clump

A 9-fold increase in FW as thin layer

B Growth as function of structure of ESM, inoculum density, flask volume and orbiting speed. Estimation of growth by settled cell volume (SCV), fresh and dry weight and image analysis.

Alayer of

Immature zygotic embryo

Extrusion of ESM (4-6 %)from the micropylar end of the megagametophyte

Proliferation as clumpson semisolid medium

B Proliferation as suspensionin liquid culture

Growth kinetics (SCV) of three lines in suspension culture (500 mg/25 ml) at 100 ml flask and 100rpm

orbiting speed

0

2

4

6

8

10

12

14

16

SCV0 SCV7 SCV14 SCV21

CULTURE DAY

GR

OW

TH (m

l)

2F471F117F11

Growth of line 2F47 in suspension culture as function of the volume flask and orbiting speed.

Inoculum 750mg/25 ml

0300600900

12001500180021002400270030003300

FW 0 FW 7 FW 14

CULTURE DAY

GR

OW

TH (m

g)

E100/50rpmE200/50rpmE100/100rpmE200/100rpm

Proliferation as a thin layer of dispersed tissue

on semisolid medium

Establishment of embryogenic lines (0.2-0.5 %)

Initiation and proliferation of ESM:Slide samples stained with 1% acetocarmine

Proliferation by division of embryonal headand formation of clusters of polarized structures

Outgrowth of ESMformation of proembryogenic and early embryogenic structures

Zygotic embryo at the cleavage polyembryony stage

and formation of clusters of polarized structures Well organized somatic embryo

Outgrowth of ESM by continuous formation of proembryogenic and early embryogenic structures

Early embryogenic structures with organized embryonal head and suspensor

Differentiation and maturation of somatic embryos:Developing SE from semisolid medium

Developing SE from liquid medium

Maturation of somatic embryos resembled that of their zygotic counterparts

Maturation of SE on gelled medium (after 16 weeksSemisolid medium: 220 embryos/g FW, from de best line and conditionLiquid medium: 44 embryos/g FW (100 ml flask/100 rpm condition)

More structured somatic embryos on solid media than in liquid suspension culture

Further development of somatic embryos requires plating the ESM from liquid to semisolid medium

Precotiledonary embryo developing the shoot apical meristem

Embryos with established polarity

Elongating cotyledonary embryos

Differentiation and maturation of somatic embryos:

Maturation of somatic embryos resembled that of their zygotic counterparts

after 16 weeks)Semisolid medium: 220 embryos/g FW, from de best line and conditionLiquid medium: 44 embryos/g FW (100 ml flask/100 rpm condition)

More structured somatic embryos on solid media than in liquid suspension culture

Further development of somatic embryos requires plating the ESM from liquid to semisolid medium

developing the shoot apical meristemElongating cotyledonary embryos Normal somatic

embryos

Germination and conversion into plants:

Acclimatized somatic plants (35%) after 2 months under greenhouse

condition

Somatic seedlings from semisolid culturedeveloped after 4 week

under germination condition (70%)

Somatic seedlings growing for 12 weeks under hydroponic culture or semisolid culture for plumule and root development (30%)

Research is in progress to improve the efficiency of plant regeneration by SE in Stone pine. Growth conditions for the establishment and maintenance of

embryogenic suspension cultures are determined to obtain normal cotiledonary somatic embryos and further conversion into viable plants

Germination and conversion into plants:

Somatic seedlings growing for 12 weeks under hydroponic culture or semisolid culture for plumule and root development (30%)

Somatic seedlings with growing radicle

15-month-old somatic plants

Research is in progress to improve the efficiency of plant regeneration by SE in Stone pine. Growth conditions for the establishment and maintenance of

embryogenic suspension cultures are determined to obtain normal cotiledonary somatic embryos and further conversion into viable plants

Preservation of embryogenic lines in liquid nitrogencultures and loose their regeneration potential. Cryoprotectant precan be optimized to survival and regrowth of ESM after thawing. In a first attempt, embryogenic lines of Stone pine were pre-treated with sorbitol-DMSO solutionimmersion in LN. All lines were recovered but underwent a large lag phase. Growth of recovered lines was comparable with the un-treated lines following 14 weeks on culture.

Actively dividing cells after 4 weeks

on culture

Initiation of polarizedstructures cleavage polyembryony

Cryopreservation:

Plantlet regeneration occurred in one of the three assayed embryogenic lines

overcome problems as maintaining for long term of cultures and loose their regeneration potential. Cryoprotectant pre-treatment and the freezing approach can be optimized to survival and regrowth of ESM after thawing. In a first attempt, embryogenic lines

DMSO solution following a slow-freezing step previous to immersion in LN. All lines were recovered but underwent a large lag phase. Growth of recovered lines

treated lines following 14 weeks on culture.

Proliferation by cleavage polyembryony

after 8 weeks on culture

Normal developed head and suspensor cells

Plantlet regeneration occurred in one of the three assayed embryogenic lines

CONCLUSIONS:

Clonal plant regeneration of Stone pine by somatic embryogenesis has been achieved

Protocols need to be improved. Current bottlenecks:

Very low frequencies of inductionCessation of growth of somatic seedlings

Clonal plant regeneration of Stone pine by somatic embryogenesis has been achieved

Protocols need to be improved. Current bottlenecks:

Very low frequencies of inductionCessation of growth of somatic seedlings

MANY THANKS FOR YOUR ATTENTION

Supported by:Prof. Luis Gil (UPM, Madrid)Dr. Javier Gordo (JCyL, Valladolid)Dr. Sven Mutke (INIA, Madrid)

MANY THANKS FOR YOUR ATTENTION

Prof. Luis Gil (UPM, Madrid)Dr. Javier Gordo (JCyL, Valladolid)Dr. Sven Mutke (INIA, Madrid)