Manihot esculenta Crantz (Cassava)

Developing

Haploid Technology

Cassava embryo sac

Development of gynogenesis in cassava for the production of doubled haploids

Zaida Lentini*1, Maria Wedzony2,

Eddie Tabares1, Maria Eugenia Buitrago1, Geraldine Restrepo1, and Freddy Vanegas1

1 Universidad Icesi, Cali, COLOMBIA 2 Pedagogical University. National Education Commission in Krakow.

Institute of Plant Physiology. Poland

Acknowledgements

This work is supported

by the Bill and Melinda Gates Foundation

through the project entitled:

Double Haploid Breeding for Cassava Enhancement

Grant # OPPGD1483

coordinated by Clair Hershey

at the International Center for Tropical Agriculture (CIAT)

Cassava

Cassava breeding is cumbersome and inefficient compared to other crops

International initiatives recognized the fundamental importance of doubled haploid (DH, plants derived from zygotic haploid cell cultures) for both plant science research and commercial success in plant breeding

DH (inbreeding) in cassava

Would create a baseline for development of homozygous germplasm for the:

identification of high-value recessive traits,

production of genetic stocks, and

application of molecular tools in breeding

Inbred progenitors would:

make breeding, maintenance, exchange, conservation and exploitation of germplasm more efficient

increase the impact of genetic transformation and the use of molecular markers, and

ease the share of genetic stocks based on botanical seed

WHY TO EVALUATE CASSAVA RESPONSE FROM OVULE /OVARY CULTURE?

• Although most use of DH technology in breeding is via anther

or microspore culture (androgenesis): Please attend to the later

talk by Prasanthi Perea S09-09 at 11:30 am, and visit Poster SP09-06 by Elzbieta Golemiec, both reporting progress of cassava. Both works are components of the Double Haploid Breeding for Cassava Enhancement Project

• Successful gynogenesis is reported in many plant species, including 21 angiosperms of economic importance (e.g.: onion,

potato, tulip, maize, sugar beet, cucumber, wheat, grape, saffron, carnation, rapeseed, rice, pearl millet, squash, mulberry, coconut, coffee, rubber tree, among others)

Why Gynogenesis?

Gynogenesis has received increased attention as

a method of choice for generating recombinant DH populations

for application in genetics (molecular mapping particularly of QTLs)

and genome sequence because of reduced genetic/ epigenetic changes

Efficient gynogenesis protocols generating large number of embryos / plants may

derive from

Culturing ovules, ovaries, or complete flower buds

Inter-specific/ inter-generic pollination between incompatible species, induces parthenogenesis in the pollinated female flower (without fecundation) generating haploid / DH embryos / plants from the ovule cultured in vitro

Main Goal

To develop an in vitro protocol for the production of doubled-haploids of cassava from cultured ovules via gynogenesis, using a reduced group of elite germplasm suitable as a model system for different ecotypes of cassava

Cassava Gynogenesis embryo formation and recovery of plants

From non-pollinated ovule cultures

From ovule cultures after inter-generic pollination with castot bean (Ricinus comunis )

Cassava Genotypes of Commercial Economic Importance in Colombia were selected

for the development of the protocol

Genotype Commercial

Name

Economic

Importance

Breeding

Importance Flowering

Months to

Fowering Origin

Adaptation

in the

Tropics

Cultivation

in Colombia

Important

Traits

HMC 1 ICA-P13 H H H 5 Colombia Medium

high altitude

Caldas,

Quindio,

Risaralda,

Valle Cauca,

Antioquia,

Tolima

High yield,

cooking

quality,

starch

MCol 1505 ICA- P12 H H H 5 Colombia

Medium

high altitude,

Subhumid

North Cost,

Tolima

Culinary

quality

M Tai 8 Corpoica-

TAI H M H 6 Thailand Sub-humid North Cost

High yield,

starch

CM 7951-5 RAYA 5 H H M 6 Colombia Acid soils

North

Cauca,

South Valle

High yield,

Good

culinary

quality

CG 489-31 Nataima 31 H H L 7 Colombia Medium

high altitude Tolima

Resistance

to White

Flies

SM 1219-9 RAYA 9 M M H 6 Colombia Acid soils North Cauca High yield

M Per 183 Peruana M M L 6 Peru Medium

high altitude

South Valle

del Cauca

Culinary

quality

TMS 60444 M Nga 11 L M H 6 Nigeria Sub-humid None

High

Somatic

Embryogen

esis

generate knowledge on cassava reproductive biology morphological characterization (histological study) of female gametophyte

embryo sac stages of development induction of embryo development

plant recovery ploidy level analysis

Gynogenesis in Cassava in vitro induction of embryo development and plant recovery

from non-pollinated female gametophyte

Ovary (3 ovules per ovary)

Open female cyathium (flower)

Pistil

Ovule Longuitunidal ovary section showing two

ovules

(A) Scheme of a cassava longitudinal cross section through stigma, stylar neck and one of three loculi of

the ovary containing an ovule

(B) Embryo sac structure (cell/ tissue) ploidy

Schemes of longitudinal cross sections of (A) pistil and (B) embryo sac

(((((sporophyte, 2n)

Cyathia are bagged prior anthesis (unpollinated / pollinated) until buds are harvested for analysis

male

female female

male are excised before bagging

Treatments at day of anthesis, 1, 2 or 3 days after anthesis

Cassava embryo sac stage of development from non-pollininated ovules at anthesis

nuclear divisions

A B

Immature embryo sac at the day of anthesis. (A) and (B) Shows the mitotic megaspore nuclear divisions to form the eight nuclei of the embryo sac at the day of anthesis. (C) Shows two polar nuclei and starch grains. Longitudinal sections of 8 µm thick were processed with a microtome and stained with Safranin-O and Fast Green. Photos were taken at 40X (A and C), and at 100X (B). PN- Polar nuclei; SG- starch grains.

PN

SG

C

micropylar end side

Chalazal end side

Cassava embryo sac at inmature stage of development from non-pollininated ovules at 1 DAA

Young cell apparatus at 1 DAA. Left, At the micropylar end of embryo sac shows the young egg cell nucleus at the same section as one synergid. Right, At the chalazal end shows a probable degeneration of the antipodal cells. Longitudinal sections of 8 µm thick were processed with a microtome and stained with Safranin-O and Fast Green. Photos were taken at 100X . EGC-Egg cell. SY-synergid, SG-Starch grains, An- Antipodal cells.

AN SG

EGC

SY

micropylar end side

Chalazal end side

Mature embryo sac apparatus organized at 2 or 3 DAA showing the egg cell, one synergid cell and starch grains at

the micropylar end side

Longitudinal sections of 8 µm thick were processed with a microtome and stained with Safranin-O and Fast Green. Photos were taken at 100X . EGC-Egg cell. SY-synergid, SG-Starch grains,

SG EGC

SY

Micropylar end side

Chalazal end side

Use of zygotic embryos as a model to establish protocol for embryo development and early rescue to

be used in gynogenesis and pollen irradiation

• Experiments were designed to define the best in vitro conditions to sustain embryo formation, its further development and growth until to recover fully developed plants

• It was decided to use in vitro culture of ovules pollinated with cassava pollen

• The objective of this study is to use it as a model for the understanding of the optimal conditions to sustain embryo development and recovery of plants, once embryo formation is induced from the non-pollinated ovules cultured in vitro

Formation of cassava zygotic embryo developed in vitro after pollination with cassava pollen

Studies used as a model to get acquainted with the

morphological changes related to cassava embryo development,

and the optimal conditions to sustain embryo development and recovery of plants,

once embryo formation is induced from the non-pollinated ovules cultured in vitro

(gynogenesis)

(A) Three-celled embryo at 7 days after pollination (DAP). (B) Pre-globular stage at 14 DAP. Longitudinal sections of 10 µm thick were processed with microtome and stained with Safranin-O and Fast Green. Photos were taken at 40X using a light microscope. ( (E) embryo, (NE) nucleated endosperm.

Histology of cassava ovules containing embryo at pre-globular stage developed in vitro

What did we learn from the formation of cassava zygotic embryo developed in vitro after

pollination with cassava pollen?

• Embryo sac is mature at 2 to 3 days after anthesis (DAA) • Embryo sac is receptive to pollination from 1 to 3 DAA • Cassava embryo develops very slowly compared to other

species • In most species a fully mature embryo (ready for

germination) is developed in 1 week after pollination • In cassava the development of an embryo at globular stage

takes about 3 weeks • Mature embryo is ready for germination 30-40 DAP

Cassava ovary / ovules in vitro culture. (A) Complete pistil showing stigma, ovary and nectar glands. (B) Ovary with excised stigma and nectar glands. (C) ovary with stigma and (D) ovary without stigma showing callus formation on the ovary wall development of unpollinated ovules after 3 weeks of culture.(E) Individual excised carpels from the ovary each containing one unpollinated ovule. (F) after 3 weeks, ovules had increased significantly in size (length and width, 1.4 fold), and protruding out of the carpel. (G) Ovule are excised from the carpel and cultured.

B

D

C

D

A

C

E

F

G

Ovule isolation and culture

Cassava Gynogenesis embryo formation and recovery of plants

From non-pollinated ovule cultures

From ovule cultures after inter-generic pollination with castot bean (Ricinus comunis )

2-cell embryo formed from non-pollinated ovules

Histology of ovules after total 6 weeks in culture.

Longitudinal sections of 4 µm each. Photos taken

at 40X.

1 – Section through 2-cellular embryo. It is accompanied by a thin leyer of central cell cytoplasm and polyploid endosperm-like nucleus is visible. For comparison see nucellar diploid nucleus pointed by arrow. 2-6. Following sections showing central cell with some more polyploid nuclei, one of them particullary large. CV – central vacuole, M – micropylar pole of ovule; N – nucellus; eln – endosperm-like nuclei. White arrow on 1B points to nucleus of nucellus, black arrows on 2 point to endosperm-like nuclei

M M

N C

CV

eln

1A 1B

M

2 3 4 5 6

eln eln

1b 2 3 1a

4

1 – Section of the embryo-sac (ES): micropylar and central part. 1a – shows a part of a globular embryo with accompanying endosperm-like nuclei (eln). 1b shows enlargement of the micropylar pole of the ES. Thin regular walls of embryonic epidermis could be seen (arrowheads). 2 – The adjacent section to the section 1. The central cell (CC) cytoplasm shows borders of the embryo proper. The two-cell supensor (S) is clearly visible. 3 – the next section show endosperm-like nuclei surrounding the embryo. Blue stained starch granules are visible in CC sytoplasm in every section. 4 – The overview of the ovule at lower magnification. Squere shows the position of ES.

eln

eln

eln

eln CC

CC CC

S

Globular embryo formed from non-pollinated ovules Histology of ovules after total 9 weeks in culture

Longitudinal sections of 4 µm each. Photos taken at 4X and 40X.

ES

Seed-like structures developed from non-pollinated isolated ovules

• Although some treatments appeared to induce embryo formation or MCS, its further development seems to be arrested

• After long term of culture, internal integument thickens significantly probably preventing nurturing the embryo under development inside the nucellus tissue

High metabolic activity in embryo sac (ES) at mycropilar (M) pole from non pollinated ovules cultured in vitro. Nucellus tissue was isolated from outer and inner integument of seed like structures (A) and cultured further. (B) Longitudinal sections of 4 µm 1-10. (P) proteins, (N) nucellus. Picture taken at 100X.

M

P P

starch

ES

Cassava Gynogenesis embryo formation and recovery of plants

From unpollinated ovule cultures

From ovule cultures after inter-generic pollination with castor bean (Ricinus comunis )

(A) Culture on MS2 medium of ovary carpels containing one ovule each. (B) Excised ovule from the carpel at 4 weeks of culture. (C) Ovule at 9 weeks of culture showing aperture at the embryo sac area. (D) Profuse induction of globular and torpedo shape embryos at 12 weeks of culture. (E) Recovery of green torpedo and cotyledonary shape embryos at 12 weeks of culture. (F) Subculture of cotyledonary embryos, and recovery of green plantlet with root and first expanded leaves. (G) Growth and development of green plants in vitro

A B C

elaiosomes

D

E

F

G

Embryo induction and recovery of green plants from in vitro cultures of ovules 2 DAP with castor bean pollen

(A) The nucellus tissue is isolated from the ovule after the rupture of the external integument. The arrow points at the multicellular structure developed inside the nucellus at the embryo sac region. (B) The callus developed inside the nucellus at the embryo sac region is isolated and cultured on a feeder layer. The arrow points at the remaining of the nucellus tissue. (C and D) Show the proliferation of embryogenic callus and differentiation of embryos. The arrows indicate the cotyledonary shape embryos. (E) Conversion of cotyledonary embryos into green plants on 4E medium.

Embryo induction and recovery of green plants from in vitro ovules cultures 2 DAP, 7 DAP or 12 DAP with castor bean pollen

Regenerated Plants

2X

2X

Embryo induction and recovery of green plants from ovules cultured in vitro 12 DAP with castor bean pollen

Direct embryo formation and profuse embryogenesis when 2,4-D is applied on stigma after pollination

Conclusions Basic steps and factors affecting gynogenesis in Cassava

Phase 1

Phase 2

Phase 3

Phase 4

decisive role

Mature embryo sac (ES) is reached 2-3 days after anthesis. ES may be receptive up to 3 days after anthesis

Ovules produced callus in liquid medium. Excised carpels containing one ovule each are cultured, then ovules are isolated 3-4 weeks after culture.

7 genotypes were selected, the most responsive so far is SM 1219-9

Picloram induces callus formation, 2,4-D or NAA induces embryo formation. Embryo induction also requires cytokinin (BAP or Kin), GA3 and high levels sucrose. Lower sucrose levels are needed for embryo maturation. Higher response is obtained in the dark

2-cell and globular embryo from non-pollinated ovules were documented at 6 -9weeks after culture

Multicellular structures from non-pollinated ovules, fully developed embryos from the embryo sac and plants from ovules pollinated with Ricinus were recovered

Conclusions • There is evidence of embryo formation from non-pollinated

ovules cultured in vitro

• Protocol for inducing embryo formation and plant regeneration from ovule culture is reproducible

• Nucellus tissue maybe isolated one month after ovule culture to stimulate further embryo development after its induction

• Flowers should be covered at least 4 days (until the stigma falls off) to avoid self pollination or outcross

• Application of 2,4-D on the stigma prior ovule culture may promote embryo formation

• IAA is currently being tested to stimulate further embryo induction and avoid callus formation

• Current experiments include the pollination with irradiated pollen to induce the formation of DH

Plant Genetic Characterization using 178 SNPs markers and flow cytometry

Analysis conducted by Luis Augusto Becerras´ group at CIAT

• Results indicate that in all the cases the plants regenerated appeared to be derived from the embryo sac

• The genetic profile of these plants confirmed their gametophyte origin

• The plants do not have a maternal genetic profile indicating that neither the integuments nor the nucellus tissue were involved in the generation of these plants

• All plants appeared to be diploid

• However, there is a variation of genome size in some of these plants respect to the cassava control genome size

Plant Genetic Characterization using 178 SNPs markers and flow cytometry (Cont.)

• Some of the plants show an increase level of homozygosity from 55% found in SM1219-9 to 70%

• This increase level of homozygosity found in these materials is interpreted to be the result of a self-pollination

• These plants are diploid based on the flow cytometry analysis, however some of them showed a reduced genome size from 50 Mb to 70 Mb respect to the control

• The haploid genome size of cassava is agreed to be between 700 to 770 Mb

• These results suggest a DNA content loss in these plants equivalent to 1 to 2 chromosomes

• These plants may be analyzed further to discard any potential misinterpretation of the data

• Another set of plants have a conspicuously different pattern with an increase level of heterozygosity interpreted to be due to outcross

• These plants are diploid based on the flow cytometry analysis and showed a genome size similar to the control

• More plants had been regenerated from the later experiments including the application of 2,4-D on the stigma prior to ovule culture and the use of IAA , which are showing to induce direct embryo induction and reducing callus formation

• Genetic analysis of these plants will be conducted shortly

Plant Genetic Characterization using 178 SNPs markers and flow cytometry (Cont.)

A Major Bottleneck

Have an ease and reliable technique to evaluate the induce

response at embryo sac after ovule culture

Poster SP09-08 Wednesday 16:00 -18:00

Optimization of histological techniques for studies of the cassava female gametophyte,

early post fertilization and embryo formation

Maria Eugenia Buitrago1, Eddie Tabares1 and Zaida Lentini*1

Thanks