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II. Broadening the Genetic Base of Cassava and Development of Interspecific Hybridization

II. Broadening the Genetic Base of Cassava and Development of Interspecific Hybridization

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CASSAVA, M. esculenta Crantz, GENETIC RESOURCES



199



maintained in the living collection at the Experimental Biology Station, Universidade de Brasília, were used for this experiment. In October 1982 the species were

planted in three rows alternated with cassava. In June 1983, 200 seeds were collected from each species and replanted in October 1984 for identification of possible natural hybridization. The following marker genes were used to identify interspecific hybrids: variegated color of fruit dominant to smooth, red color of flower

disk dominant to yellow, setaceous bracteole dominant to foliaceous, and noded

stem dominant to smooth. Observations of growth habit, height, stem texture, and

tuber formation were also recorded. In addition to open pollination for the previously mentioned species, 400 manual crosses with pollen of cassava cultivar Catelo were realized. Of 200 seeds of M. neusana, only 43 seedlings emerged, of which

2 hybrids were identified. Interspecific hybrids were identified by dominant markers from cassava; noded stem, setaceous bracteoles, ribbed fruit, and tuberculated

root (Table XII). Other characters provided indirect evidence of hybridization. The

200 seeds collected from M. anomala gave rise to 112 seedling. Of these, 3

seedlings showed characteristics of interspecific hybridization. Only 1 seedling survived to maturity. This hybrid plant exhibited dominant phenotypes from cassava,

namely, ribbed fruit, red color of the flower disk, noded stem, and tuberous root

(Table XII). These results show that glabrous stem, setaceous–foliaceous bracteoles, red-creamy color of flower disks, variegated-green color of fruit, and ribbed–

nonribbed fruit are simple marker genes that can be used to recognize interspecific hybridization. It is evident that interspecific barriers between Manihot species

can be broken by the use of an abundant diversity of pollinator gametes transmitted by insect vectors. Interspecific crosses were difficult to fertilize manually in the

Table XII

Comparison of Morphological Characteristics of M. neusana, Cassava, and Their Hybrid

Characteristic



M. neusana



Cassava



Hybrid



Growth habit

Young stem texture

Bracteoles

Fruits



Erect shrub (1.5–2 m)

Glabrous

Setaceous

Ovoid, ribbed, green



Tuber formation



Procumbent shrub (1.5–2 m)

Hairy

Foliaceous

Globose, without

ribs, variegated

None



Forms tubers



Erect shrub (1.5–2 m)

Hairy

Setaceous

Ovoid, ribbed,

variegated

Forms tubers



Growth habit

Young stem texture

Bracteoles

Flower disk color

Leaf form

Fruit

Tuber formation



Erect shrub (2–2.5 m)

Hairy

Semifoliaceous

Creamy

Anomala

Globose, without ribs

Scarely forms tubers



Erect shrub (1.5–2 m)

Glabrous

Setaceous

Red

Lobed; 5 lobes

Ovoid, ribbed

Forms tubers



Erect shrub (1.5–2 m)

Hairy

Setaceous

Red

Anomala

Ovoid, ribbed

Forms tubers



200



NAGIB M. A. NASSAR



present and in previous crosses (Nassar, 1980a). The evidence suggests that barriers between cassava and other Manihot species are weak and recently evolved. It

seems they have arisen not as a primary isolating event but rather secondarily after

geographic isolation. Nassar (1978b) postulated that cassava is an interspecific hybrid that appeared by domestication approximately 2000 years ago or less.



B. DEVELOPMENT OF CASSAVA INTERSPECIFIC HYBRIDS

The wild Manihot species of M. neusana Nassar was hybridized with the cassava clone Catelo through controlled hybridization with the help of pollinating insects

(Nassar, 1989). An interspecific hybrid that combined marker genes of both parents

was obtained. The marker genes were ribbed fruit, acquired from cassava, and variegated fruit color from M. neusana. This hybrid (HN) was backcrossed with cassava and used as a pollinator in the first trial and as a fruit carrier in the second trial.

Seeds were obtained from both crosses, but only one plant could be raised from each;

HO1 was the result of the interspecific hybrid (HN) as maternal plant (seed carrier),

and HO4 resulted from crosses in which the interspecific hybrid (HN) was used as

pollinator. The three hybrid plants (HN, H1, and H4) were studied cytogenetically

for both meiotic and mitotic behavior. For the study of meiosis, inflorescences were

fixed in a mixture of three parts absolute alcohol and one part glacial acetic acid and

kept in a refrigerator for 24 h. The anthers were smeared with acetic carmine. Chromosome configurations in the metaphase, chromosome distribution in anaphase I,

and tetrad formation were also studied. Pollen viability had been determined by using acetocarmine and iodine stain (Nassar, 1978a). For the mitotic study, root tips

were left in 0.2% colchicine for 2 h and then fixed in acetic alcohol for 24 h. Before

smearing with acetocarmine, they were treated with 1 N HCl for 10 min.

1. Meiotic Behavior of the F1 Hybrids (HN)

One hundred pollen mother cells (PMCs) were studied in metaphase I of the interspecific hybrid M. neusana with cassava; 30 PMCs in metaphase II and 1000

tetrads of the same material were also investigated. The study of metaphase I

showed different chromosome configurations, as shown in Table XIII. The average bivalent frequency in all cells of metaphase I was 16.13 per cell. The high frequency of univalents was attributed to lack of synapses between chromosomes or

failure of the two species to remain associated. Virtually the only other report on

this subject is that of Magoon et al. (1970), in which chromosome pairing in the

interspecific hybrid M. glaziovii (rubber tree) and cassava was studied, and a regular synapsis led the authors to conclude that there is a strong relationship between

this species and cassava. Nassar et al. (1986) suggested that the material used by

Magoon et al. was not a pure M. glaziovii but rather a natural interspecific hybrid

between this species and cassava. If this is true, the supposed interspecific hybrid



CASSAVA, M. esculenta Crantz, GENETIC RESOURCES



201



Table XIII

Frequency of Chromosome Configuration of Metaphase I in Interspecific

Manihot Hybrids and Their Parents



M. neusana

Cassava

GN

HO1

HO4



PCMs (No.)



Trivalents



Mean

Bivalents



Univalents



20

20

100

30

100









1.86

1.63



18.00

18.00

17.00

16.13

12.41







1.58

0.13

8.84



would be a backcrossed progeny. The study of anaphase I showed that of 40 PMCs

studied, 38 cells exhibited laggards, which were attributed to the occurrence of

univalents resulting from nonhomologous chromosomes.

Anaphase II showed meiotic restitution. Of 33 PMCs studied in this phase, 5

cells exhibited a second meiotic restitution (SMR), forming 36 chromosomes on

each pole. Apparently this phenomenon is a consequence of meiotic disturbance

in the hybrid. An example of this disturbance was the breakdown of anaphase I.

This was probably due to disharmony between the two different genomes. Nassar

(1991) documented this phenomenon in the interspecific hybrids of cassava with

M. pseudoglaziovii. The presence of such restitution was confirmed in the following tetrad stage, in which the formation of both dyads and tetrads was observed.

In various crops, interspecific hybridization has led to the disturbance of meiotic division, with consequent meiotic restitution, e.g., in Trifolium pratense by

Parrot and Smith (1984) and in Medicago spp. by Vorsa and Bingham (1979). In

manioc species, Hahn et al. (1990) reported 2n pollen formation in wild species in

addition to certain clones of cassava. The detection of this phenomenon enabled

these researchers to isolate triploid and tetraploid types from progeny that came

from crosses of cassava with certain wild Manihot species, namely, M. glaziovii

and M. epruinosa. These types proved much more productive than commercial

clones used in Nigeria. Nassar (1991) manipulated the meiotic restitution occurring in interspecific hybrids of M. pseudoglaziovii with cassava to produce triploid

types that showed very good productivity under semiarid conditions. The discovery of the frequent occurrence of this phenomenon in interspecific hybrids of cassava offers an effective tool for the production of polyploid types by sexual means

instead of the traditional method of colchicine applied to vegetative parts, which

normally induces unstable, chimeral types (Abraham et al., 1964). An additional

advantage is that production of triploid types may lead to production of trisomics

among their progeny. If genes which control productivity in cassava are polygenes

with addictive model action, as is the case for many crops, certain trissomics of

this crop may be more productive than their diploid ancestors. In general, the pro-



202



NAGIB M. A. NASSAR



duction of polyploidy via sexual means is advantageous from both genetic and

evolution standpoints because it offers a vigorous heterotic effect and releases useful genetic variability adaptations.

For the study of the tetrad stage, 1065 PMCs were investigated. Of these, 62 cells

formed dyads and 62 cells formed micronuclei. The presence of dyads and tryads at

this stage confirms that observed earlier in the anaphase: the occurrence of first- and

second-divisioin meiotic restitution (FMR and SMR). Both types are capable of producing 2n gametes. However, the FMR is more valuable than the SMR since it preserves the major part of its heterosis and epistatic interaction (Mendiburu and Peloquin, 1977; Parrot and Smith, 1983; Vorsa and Bingham, 1979).

2. Cytogenetic Behavior of the Backcrossed Generation (HO1)

Table XIV shows the frequency of chromosome configurations at metaphase I.

Bivalents, trivalents and univalents were present, with a mean of 16.1 bivalents,

1.86 trivalents, and 0.13 univalents. The presence to trivalents indicated aneuploidy in this hybrid. This was confirmed by mitotic counting, which showed 2n

ϭ 38, i.e., 2n ϩ 2. In anaphase I, the presence of laggards with different frequencies was recorded. The study of 900 tetrads showed 644 normal ones, 218 micronuclei, 12 dyads, and 26 tryads. Analysis of pollen viability revealed that only

35.8% were viable (Table XV).

3. Cytogenetic Behavior of the Backcrossed Generation (HO4)

One hundred PMCs at metaphase I were studied. Again, bivalents, trivalents,

and univalents with averages of 12.4, 1.6, and 8.8, respectively, were observed.

The total chromosome count for the different configurations was 38. This showed

a constitution of 2n ϩ 1 ϩ 1, which was confirmed by root tip mitotic counting.

In anaphase I, of 32 PMCs studied, 31 proved to have laggards. In anaphase II, 35

PMCs were examined; of these, 7 cells appeared as restitution nuclei, and this was

later confirmed in the tetrad stage. In the tetrad stage, 1196 sporocytes were obTable XIV

Diploid Pollen in Interspecific Manihot Hybrids and Their Parents

Diploid pollen



M. neusana

Cassava

HN

HO1

HO4



Pollen grains

examined (No.)



No.



%



818

1162

1235

1128

1007



3

3

20

8

6



0.36

0.26

1.62

0.71

0.60



203



CASSAVA, M. esculenta Crantz, GENETIC RESOURCES

Table XV

Viability of Pollen of the Interspecific Manihot Hybrids and Their Parents

Viable pollen

Pollen

analyzed (No.)



No.



%



Nonviable

pollen



1001

1235

1830

1542



818

1162

655

273



81.72

94.09

35.80

17.70



183

73

1175

1269



M. neusana

HN

HO1

HO4



served. Of these, 326 were normal, 826 contained micronuclei, 25 were tryads, and

19 were dyads. The study of pollen viability showed a very low viable grain percentage of 17.7% (Table XV).

4. Cytogenetic Behavior of the Parents

The cassava clone EB 01 showed a regular meiotic division in all of the 20

PMCs studied, with 18 bivalents formed. Of the 950 tetrads that were examined,

942 were normal, 5 contained micronuclei, and 3 were tryads (Table XVI). The

study in which M. neusana was used as a parent showed it to have a regular meiosis with 18 bivalent formations. Of 1011 tetrads studied, 1003 were normal, whereas 6 had micronuclei and 2 were dyads. The pollen viability was 81.72% (Table

XV). Mitotic counting of root tips showed 2n ϭ 36. This was the first report of a

chromosome number for this new species described by Nassar (1985).

5. Evolutionary and Plant Breeding Significance

The fertility of the hybrid HO1 indicates the possibility of further manipulation

of this hybrid through backcrosses to transfer useful genes of M. neusana to cassava. The backcrossed generations produced were aneuploid 2n ϩ 1 ϩ 1 in both

Table XVI

Analysis of Tetrads of Interspecific Manihot Hybrids and Their Parents

Tetrads



M. neusana

Cassava

HN

HO1



PMCs



Tryads



Dyads



Total



Normal



%



No.



%



No.



%



No.



%



1011

9502

1065

900



1003

942

694

644



99.22

99.15

65.15

71.55



6

5

262

218



0.59

0.53

24.60

24.22



2

3

62

26



0.19

0.31

5.82

2.88







47

12







4.41

1.33



204



NAGIB M. A. NASSAR



cases studied (HO1 and HO4). In the case of hybrid HO4, the plant was completely

sterile, having a chromosomal constitution of 2n ϩ 1 ϩ 1. Obviously, this hybrid

resulted from fertilization of a pollen gamete n ϩ 1 ϩ 1 of the parent hybrid (HN),

with a cassava ovule of “n.” On the other hand, when the interspecific hybrid HN

was used as a maternal plant, a fertile progeny was obtained. When it was used as

a pollen parent in the backcross with cassava, it resulted in the production of a sterile progeny (HO4). This was probably due to the elimination of fertile embryo

genotypes in the progeny because of incompatibility or disharmony between them

and the endosperm.

The partial fertility of the backcrossed generation (HO1) shows that the species

M. neusana may be classified within the secondary gene pool of cassava according to the concept of Harlan and de Wet (1971). Other Manihot species that may

be included in this category are M. melanobasis (Jennings, 1959), M. glaziovii

(Magoon, 1970), M. reptans, M. zenhtneri, M. anomala, M. oligantha, M. pohlii

(Nassar et al., 1986), and M. dichotoma, M. epruinosa, and M. leptophylla (Hahn

et al., 1990). It was concluded that the cassava hybrid with M. neusana showed irregular meiotic behavior in the lack of complete chromosome pairing, formation

of univalents in metaphase I, chromosome retardation in anaphase I, micronuclei

in the tetrad, and meiotic restitution. When backcrossed to cassava, the interspecific hybrid produced two aneuploids 2n ϩ 1 ϩ 1. These showed irregular meiosis, partial chromosome pairing, and the presence of meiotic restitution. The two

backcrossed F2 hybrids differed in regard to pollen-viable genetic constitutions.

The meiotic restitution continued to occur in F2 hybrids, which showed that it must

be correlated with interspecific meiotic irregularity.



C. DEVELOPMENT OF CASSAVA INTERSPECIFIC HYBRIDS

FOR SAVANNA (CERRADO) CONDITIONS

Interspecific hybrids of cassava with M. glaziovii and M. pseudoglaziovii were

produced by Nassar in 1991 (Nassar et al., 1996a). They were propagated by cuttings and planted in November 1992 alternately with clone Sonora. This clone was

chosen because of its high resistance to bacteriose. Seeds were collected from cassava and planted in November 1993. Out of 182 seeds whole plants. In March

1994, these plants were reproduced by cuttings; six of each were planted for evaluation of productivity and survival during the drought season of June to October

(5 months). In November 1994, plants which survived were evaluated for root formation. Ten clones were selected and given to the Semi Arid Centre at Pernambuco for propagation and cultivation under semiarid conditions of northeastern

Brazil. The selected clones were characterized morphologically according to

Rogers and Appan (1973) and Nassar and Grattapaglia (1986). This characterization was aimed at detecting the association of different morphological characters



CASSAVA, M. esculenta Crantz, GENETIC RESOURCES



205



with tolerance to drought. Since polyploidy offers another alternative for this tolerance, the previously mentioned interspecific hybrids and several others available

in our living collection were investigated cytologically to detect formation of diads and triads as evidence of unreduced 2n microspore production (Vasquez and

Nassar, 1994). This will enable the selection of possible progenitors of polyploid

types.

For cytogenetic study, 10 interspecific hybrids and/or their progenies were used:

F1 M. glaziovii ϫ cassava (3 genotypes), F2 M. epruinosa ϫ cassava, F2 M. anomala ϫ cassava (3 genotypes), F3 M. pseudoglaciovvi ϫ cassava (2 genotypes), and

F4 M. pseudoglaciovvi ϫ cassava.

Chromosome association in meiotic metaphase I and the occurrence of dyads,

tryads, and pollen viability were studied. For the meiotic study, inflorescence was

fixed in a 3:1 mixture of absolute alcohol and glacial acetic acid and kept at 5ЊC

for 24 h. The anthers were smeared in acetocarmine stain. Chromosome configurations at metaphase I and sporad formation were studied. For the pollen viability

study, one or two flowers per plant were selected, and their pollen was crushed

from anthers in acetocarmine preparations and scanned for viability. Pollen counts

and percentage of stained normal pollen were calculated.

Of the 182 seeds planted, only 35 germinated, established, and developed to the

flowering phase. This is due to dormancy of wild seed retained in F2 progeny.

These 35 plants were multiplied by cuttings to evaluate their performance for root

production. Only those plants which gave more than 2-kg roots with a medium 2.3

kg per plant by 8 months were selected for multiplication for further cultivation.

Morphological characterization showed that certain characters were associated

with tolerance to drought. As seen in Tables XVII and XVIII, all selected clones

have a notable brown, thick, and rough superficial epiderm. It seems that the

brown-colored thick epiderm is associated with tolerance to drought because of its

isolative nature, which impedes evaporation. All the wild species investigated by

the author have fibrous roots with brown external color and their epidermic layer

is thick. It is believed, therefore, that this character is inherited from the wild.

Graner (1942) reported that this character behaves dominant to white. Anatomically, the distinct portion of the enlarged root is composed of three sections. First,

a layer referred to as the phelloderm which is generally composed of the previously mentioned epidermis, a subepidermis, and a thicker inner layer. The phelloderm is thick and easily separated from the next inner layer. Second, a layer of

parenchymatous cells that constitutes the bulk of the root and is the carbohydrate

storage region. Third, a portion called the cortex of flesh at the center of the root

is a well-defined central vascular core. As noted previously, the outer epidermis is

so fine that it is difficult to measure, but it is possible to evaluate its thickness using the naked eye. It is about 0.5 mm in the thickest types.

The second interesting case in selected clones is the prominence of leaf scars on

stems. All selected clones have a prominent enlarged leaf scar. This character



Table XVII

Characters Used in Clone Characterization

Root characters

1. Surface of root

A. Smooth

B. Rough

2. Thickness of epiderm layer

A. Thick (>. 0.3 mm)

B. Thin (<. 0.3 mm)

3. External color of root

A. Light brown-yellow

B. Brown, dark brown, reddish brown

C. Light brown, tan, light tan

D. Pinkish brown, pinkish tan

E. Pinkish white, light pink, pink

4. Root flesh color

A. White to cream

B. White to cream with pink

C. Cream-yellow to yellow

D. Cream-yellow to yellow with pink

5. Color of stem

A. Silver

B. Silver-brown

C. Brown

D. Yellow

6. Nature of scars on stem

A. Smooth

B. Slightly raised

C. Moderately raised

D. Very large

7. Branching of plant

A. One branch at top or no branches

B. One or two branches but not one branch at top

C. More than two branches

Leaf characters

8. Leaf lobe shape

A. Obovate

B. Linear

9. Sinuosity of leaf lobes

A. Pandurate

B. Some sinuosity

C. Simple (not sinuous)

D. Logical (linear)

10. Petiole color

A. Red

B. Greenish red

C. Reddish green

D. Green

11. Color of young foliage

A. Reddish blue

B. Bluish green

C. Green



207



CASSAVA, M. esculenta Crantz, GENETIC RESOURCES

Table XVIII

Morphological Characteristics of Selected Clones

Characteristic



1



2



3



4



5



6



8



9



10



11



1. Surface of root

2. Thickness of epiderm layer

3. External color of root

4. Root flesh color

5. Color of stem

6. Nature of scars on stem

8. Leaf lobe shape

9. Sinuosity of leaf lobes

10. Petiole color

11. Color of young foliage



B

A

B

A

B

C

B

A

B

C



B

A

B

B

B

C

A

A

D

C



B

A

B

B

B

D

A

A

A

A



B

A

B

C

B

C

A

D

A

C



B

A

B

A

B

C

B

A

A

A



B

A

B

B

B

C

A

A

C

A



B

A

B

A

B

C

A

C

D

C



B

A

B

A

B

C

B

B

B

B



B

A

B

A

B

C

A

A

B

C



B

A

B

A

B

D

A

D

B

B



Note. Numbers and letters correlate with those used in Table XVII.



which seems to be well associated with enlarged root formation in the hybrid progeny, apparently came from cassava. All wild species studied by the author have a

smooth stem without any leaf scar. All selected clones gave deep fibrous roots in

addition to enlarged ones. It seems that this character is inherited from the wild.

This appears to be a mechanism of cassavas to tolerate drought since they are capable of capturing water from long distance. Both wild species and their interspecific hybrids produced long, deep roots from the fourth month onwards, reaching

4 or 5 m when the plants were 1 year old.

Another mechanism of tolerance to drought is the thick epiderm layer, probably because of its structure, it impedes evaporation. The dieback of the vegetative

parts to the crown in dry season was the third common character shown by all the

selected clones. Presumably this habit helps plants to reduce respiration and consumption of carbohydrate deposits. From this study, it is obvious that breeders can

make use of morphological characterization as a criterion to detect the association

of morphological characters and drought tolerance and consequently selection of

genotypes complying with this objective.



D. OVERCOMING CROSSING BARRIERS BETWEEN

CASSAVA, M. esculenta Crantz,

AND A WILD RELATIVE, M. pohlii WARWA

Manihot pohlii has a high resistance to stem borers, Coelosternus spp., and bacteria Xanthomonas Manihotis. Hybrids of M. pohlii and other species with cassava can be obtained but only at a very low frequency due to interspecific crossing

barriers (Nassar et al., 1986). However, one technique for overcoming such barri-



208



NAGIB M. A. NASSAR



ers involves the use of pollen mixes which combine the pollen intended for syngamy with “mentor” pollen of the maternal species. This technique has been reported for many plant taxa, including Populus (Knox et al., 1972), Malus (Dayton, 1974), Cosmos (Howlett et al., 1975), and Petunia (Sastri and Shivanna,

1976). The role of the mentor pollen is to facilitate fertilization by foreign pollen.

Apparently, the mentor pollen supplies proteinaceous substances which permit the

foreign incompatible pollen grains to germinate (Knox et al., 1972). A treatment

destroys the generative function of the pollen grain without affecting germination

and growth of the pollen tube and consequently does not affect stimulation. The

stimulating effect of destroyed pollen is due to protein recognition substances.

They are liberated by pollen grains while germinating and have enzymatic and

antigenic properties. These substances are localized in the internal layer of the

pollen surface and are correlated with pollen germination and growth of its tube

on the stigma surface. They have been localized by cytochemical means in the cellulose intine (Knox et al., 1972). Many studies using this technique have been

successful in overcoming an incompatibility barrier (Brewer and Henstra, 1974;

Williams and Church, 1975). Subsequent work indicated the need to refine the

preparation of the mentor pollen by using freeze–thaw cycles or methanol treatment (Knox et al., 1972). Nassar et al. (1996b) successfully used freezed mentor

pollen in hybridizing cassava with M. pohlii. In addition to the mentor effect, other techniques seem to have potential for overcoming interspecific barriers. One of

these may be the use of a bridge species. Nassar et al., inspired by the capacity of

M. neusana Nassar to cross easily with all Manihot species growing in the vicinity, used it as a bridge species. Very little information is available regarding the

bridge technique. Probably the only case is that of Dionne (1963), who used

Solanum acaule Bitter as a bridge between Solanum tuserosum L. and Solanum

bulbocostanum Dunal.

A natural hybrid between M. pohlii and M. neusana Nassar was used by Nassar

et al. (1996b) as a bridge species. The hybrid was identified by fruit marker genes,

which produce variegation of fruit color in M. neusana and a straight white line in

M. pohlii fruit. Crosses of this hybrid (named HNP) and cassava (clone EB 05)

were carried out from January 1993 to January 1994. Flowers were taped shut for

2 days until they had been pollinated manually. Pollination of both the hybrid HNP

and cassava was done with pollen mixes of cassava and M. pohlii. Manihot pohlii

pollen used as mentor pollen was sucessively frozen for 5 min at Ϫ4ЊC and thawed

for 30 min for a period of 105 min. The purpose of this treatment was to kill the

mentor pollen and increase the chance of obtaining interspecific hybrid seed. To

verify the presence of any autoincompatibility that would interfere in the controlled crosses in M. polii and cassava, a controlled autopollination was undertaken. Crosses between cassava and M. pohlii (POH) were carried out using mentor

pollen in one trial and untreated pollen in the second trial. Seeds were collected

from both crosses and planted during the next growing season.



CASSAVA, M. esculenta Crantz, GENETIC RESOURCES



209



The pollination of M. pohlii by untreated cassava pollen did not produce any

fruit set (Table XIX), whereas crosses with mixed pollen of cassava and mentor

resulted in the production of 21 seeds, which is 4.9% of the possible maximum

(assuming that every fruit has three ovules). Using cassava as a maternal parent,

pollination by untreated pollen M. pohlii did not result in any seed. This clearly

demonstrates the effect of mentor pollen in the crosses of cassava with M. pohlii.

It likewise means that the freeze–thawing treatment administered to M. pohlii

pollen, although it did kill the pollen, did not affect its stimulatory function so that

all the seed produced by the mentor effect had embryos and endosperm. This result indicates that only the stigmatic barrier functions in preventing crossing in

these species.

Postfertilization mechanisms fail to prevent crossing. Only one plant germiseeds to severe dormancy. This plant bore fruits carrying the marker genes of

both M. pohlii and cassava: a straight white line from M. pohlii and winged fruit

from cassava. The mentor effect has also been successfully used in Populus and

Cosmos. In these genera, interspecific incompatibilities have been overcome by

using compatible but dead pollen (Knox et al., 1972). These studies suggest that

this phenomenon is due to the release of proteinaceous recognitioin factors from

the wall of the killed compatible pollen, masking the rejection reaction of the recipient stigma. Nassar et al.’s report (1996b) represents the first case of obtaining

hybrid seed of M. pohlii and cassava and its further reproduction. Despite the useful characters of M. pohlii, no successful breeding program has been carried out

due to a lack of hybrids between this species and cassava.

The use of M. neusana as a bridge species through the hybrid M. neusana–M.

pohlii has improved seed set. When used as a maternal parent pollinated by cassava, it gave seed in 3.5% of cases, whereas the reciprocal crosses had greatly improved seed set, yielding 25.9% (Table XX). The combined treatment of both

species (bridge and mentor) produced 3.5% seed production. The success of M.

neusana as a bridge species between M. pohlii and cassava may occur because the

two genomes of M. neusana carry different genetic mechanisms of cross incompatibility. This hypothesis is confirmed by observations on crossing behavior of

this species in the living collection of wild Manihot species. Manihot neusana has

Table XIX

Crosses Attempted between M. pohlii and M. esculenta (Cassava) and Numbers of Fruit

and Seed Produced

Treatmenta



Flowers pollinated



No. Fruit



No. Seed



145

142



0

10



0

21



1. Cassava ϫ M. pohlii

2. M. pohlii ϫ cassava

a1,



Without mentor; 2, with mentor.



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II. Broadening the Genetic Base of Cassava and Development of Interspecific Hybridization

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