209 Ali et al.

Int. J. Biosci. 2015

RESEARCH PAPER OPEN ACCESS

Development and cytogenetic characterization of some

interspecific crosses in rice (Oryza sativa L. × Oryza

rufipogon Griff.)

Hamid Ali1*, Fida Muhammad Abbasi1, Habib Ahmad1, Muhammad Abid Khan2,

Hidayat Ullah1, Syyed Gauhar Jamal1, Abdul Waheed1, Shujaul Mulk Khan2,

Muniba Fida Abbasi1

1Department of Genetics, Hazara University Mansehra, Pakistan

2Department of Botany, Hazara University Mansehra, Pakistan

Key words: Oryza sativa, Oryza rufipogon, Wide Hybridization, Cross ability.

http://dx.doi.org/10.12692/ijb/6.5.209-219 Article published on March 14, 2015

Abstract

Oryza rufipogon Griff., is a valuable source of resistant genes to various biotic and abiotic stresses. These

resistant genes can be easily transferred to cultivated rice (Oryza sativa L.), through wide hybridization.

Therefore, the present study was carried out to evaluate interspecific crosses between O. sativa cv. Bas-385, IR-

6, KS-282 and O. rufipogon (Acc. 106516). Immature spikelets of cultivated varieties (♀) were emasculated and

shed with sufficient pollens of O. rufipogon (♂). The pollinated spikelets were sprayed immediately with 75 ppm

GA3 to enhance pollen germination. A mixture of 100 ppm GA3 and 25 ppm NAA was then applied twice daily for

5 consecutive days to prevent embryo abortion. The cross seeds (F0) were harvested 30 days after pollination.

Seed setting rate and cross ability was determined for each cross combination. The cross ability between O.

sativa and O. rufipogon varied from 4.35-8.33 (%). The highest cross ability was observed in IR-6 × O.

rufipogon (8.33%), followed by Bas-385 × O. rufipogon (6.39%) and KS-282 × O. rufipogon (4.35%). The hybrid

plants were intermediate between the two parents in phenotypes such as basal leaf sheath color but there was an

overall dominance of wild traits in the F1 hybrids. In order to assess meiotic affinity between O. sativa and O.

rufipogon, chromosome behaviors in pollen mother cells (PMCs) of parents and their F1 hybrids were analyzed.

The frequencies of abnormalities such as univalents (I), trivalents (III) and laggards were slightly higher in all

hybrids at metaphase-I. Despite the low occurrence of such abnormalities, on the average all hybrids showed

normal meiosis with remarkably, high degree of chromosome pairing. At metaphase-I, all hybrids had more than

10.96 bivalents and 21.78 chiasma/PMC. Thus favorable alleles can be easily transferred from O. rufipogon to

cultivated rice.

* Corresponding Author: Hamid Ali biotechd.ali@gmail.com

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print), 2222-5234 (Online)

http://www.innspub.net

Vol. 6, No. 5, p. 209-219, 2015

210 Ali et al.

Int. J. Biosci. 2015

Introduction

Improvement of rice with respect to resistance to

biotic and abiotic stresses through introgression of

alien genetic variation is an important breeding

objective of wide hybridization (Mandal and Gupta,

1997; Zhou et al., 2008). Wide hybridization and

chromosome manipulation are the important

techniques to transfer useful genes from wild species

to cultivated rice, Oryza sativa L. Following these

methods, several genetic and cytogenetic stocks such

as monosomic alien addition lines (MAALs),

introgression lines (ILs) and mapping population

have been developed in rice (Brar and Khush, 1997).

Introgressive hybridization can often lead to rapid

genomic changes, including chromosomal

rearrangements, genome expansion, differential gene

expression and gene silencing, some of which are

mediated by transposable elements (Baack and

Rieseberg, 2007). These genomic changes may lead to

beneficial new phenotypes and selection for fertility

and ecological traits may in turn alter genome

structure (Baack and Rieseberg, 2007). Knowledge of

the cytogenetic relationships between cultivated

species and their wild relatives has still greater scope

to transfer alien genes of interest. Several innovative

approaches in breeding crops have been developed

and one of them demands constant reference to the

chromosomal status of the breeding materials. The

chromosomal status and ploidy level of species in

gene introgressive breeding program ultimately

depends on the meiotic behavior at different stages of

meiosis (Sinha et al., 1983). Moreover, cytogenetic

basis of genome and evolutionary analyses are crucial

for directing our efforts to search for beneficial gene/s

in wild species of rice. Although landraces and wild

species are in general agronomically inferior to

cultivated rice, the transfer of favorable alleles for

disease resistance, tolerance to abiotic stresses,

agronomic traits such as yield heterosis, higher

protein quantity and other quality related traits are

still feasible (Brar and Khush, 1997; Fu et al., 2008;

McCouch et al., 2007). Oryza rufipogon Griff.

is a member of more than 20 wild species in genus

Oryza (Vaughan 1989), and it is commonly regarded

as the wild progenitor of Asian cultivated rice, O.

sativa, which is further subdivided into two

subspecies, ssp. indica and ssp. japonica (Cheng et al.,

2003; Londo et al., 2006). It is perennial,

photoperiod sensitive, largely cross-fertilized, and

widely distributed from southern China, south and

Southeast Asia to Papua New Guinea and northern

Australia. It grows in areas with year round water,

such as swamps and lakes. Because of their

important role in providing beneficial genes for rice

breeding (Khush, 1997; Vaughan et al., 2003; Zhang

et al., 2006; Xie et al., 2010), O. rufipogon has long

been the subject to extensive taxonomic, phylogenetic

and population studies using a variety of approaches.

However, introgression of these genes involving

Pakistani rice varieties is still not fully investigated.

Present study was therefore conducted to produce F1

hybrids of O. rufipogon and O. sativa (Pakistani

cultivars). Meiotic chromosome analysis experiments

were conducted to assess genomic affinity as a

prerequisite of alien gene transfer.

Materials and methods

Plant material

Three widely grown Pakistani commercial cultivars

viz. IR-6, KS-282 and Bas-385 (O. sativa L. sub

species indica) and common wild rice O. rufipogon

Griff. (Acc. No. 106516) were used as parental

materials in the present study. Seeds of cultivated

varieties were obtained from the Gene Bank of Plant

Genetic Resource Institution (PGRI), NARC (National

Agriculture Research Centre), Islamabad, Pakistan.

Seeds of O. rufipogon were obtained from the

International Rice Germplasm Centre (IRGC), of the

International Rice Research Institute (IRRI),

Philippines.

Seed dormancy of O. rufipogon was broken through

dry heat treatment at 50° C for 7 days and hull

removal. Seeds of wild species as well as cultivated

varieties were pregerminated at 33° C in sterilized

petri dishes lined with filter paper. The germinated

seedlings were transferred to plastic pots and grown

under standard agronomic conditions. The present

study was conducted during rice growing seasons of

2008 & 2009, in the Department of Genetics, Hazara

211 Ali et al.

Int. J. Biosci. 2015

University, Mansehra Pakistan.

Production of interspecific hybrids (O. sativa × O.

rufipogon)

Inter specific crosses were made between cultivated

varieties viz. Bas-385, IR-6 and KSK-282 and wild

rice O. rufipogon (IRGC Acc. No. 106516). Cultivated

varieties were used as seed parents (♀), while O.

rufipogon (♂) was used as pollinator. To prepare

female parents, panicles of rice plants at appropriate

stage (when ¼ th of panicle had emerged from the

flag leaf) were emasculated and pollinated with

sufficient pollen of wild species on following day.

Gibberellic acid (GA3) 75 ppm was sprayed onto

pollinated panicles immediately after pollination. The

pollinated panicles were also sprayed twice a day for

five days with a mixture of growth hormones GA3 and

NAA (Naphthalene acetic acid) in the proportion of

100 and 25 mg L-1, respectively, beginning with the

afternoon following pollination. The pollinated

panicles were harvested 30 days after pollination, F0

seeds were collected and % seed set was determined

for each cross as reported by Guo et al., 2009.

For confirmation of interspecific crosses, the F0 seeds

were grown in pots. The F1 hybrids obtained were

confirmed through morphological markers such as

basal leaf sheath color. Phenotypic data was recorded

from all the flowering tillers of each plant at

appropriate stage of growth using IRRI standard

descriptor, Technical Bulletin 4, 1980.

Meiotic chromosomes analysis

The sporocytes of the parents and F1 hybrids were

collected at the early booting stage between 7.00-

9.00 a.m. These were fixed in Farmer’s solution (3

parts of 95% ethyl alcohol +1 part of glacial acetic

acid) for 24 hours at room temperature. Traces of

ferric chloride were added as mordant. Pollen

mother cells (PMCs) smears were prepared by

squashing the anthers in 2 % acetocarmine. Data

on chromosome association such as univalents,

bivalents or other configuration at

diakinesis/metaphase-I and distribution of

chromosomes at anaphase-I was recorded.

Results

Production and confirmation of interspecific crosses

Interspecific crosses were made between cultivated

varieties and wild species of rice during the present

investigation. A total of 624, 460 and 360 spikelet of

cultivated varieties IR-6, KS-282 and Bas-385 were

pollinated, respectively. The percentage of seed set

was 19.7, 17.3 and 16.3 (%) for IR-6 × O. rufipogon,

KS-282 × O. rufipogon and Bas-385 × O. rufipogon,

respectively. Whereas, rate of germination was 42.28,

25.12 and 39.20 (%) for IR-6 × O. rufipogon, KS-282

× O. rufipogon and Bas-385 × O. rufipogon,

respectively (Table 1).

Table 1. Seed setting rate and cross ability of interspecific crosses of O. sativa and O. rufipogon.

Cross

combination

Spikelet pollinated (No) Seed set (%) Seed germination (%) F1 hybrids produced (No) Cross ability (%)

IR-6 × O.

rufipogon

624 19.7 42.28 52 8.33

KS-282 × O.

rufipogon

460 17.3 25.12 20 4.35

Bas-385 × O.

rufipogon

360 16.3 39.20 23 6.39

The cross ability between O. sativa and O. rufipogon

varied from 4.35-8.33 (%). The highest cross ability

was observed in IR-6 × O. rufipogon (8.33%) and

least in KS-282 × O. rufipogon (4.35%). Based on

cross ability, IR-6 showed a close affinity with O.

rufipogon (Table 1).

The hybrid plants were intermediate between two

parents in phenotypes such as basal leaf sheath color,

stigma color and awn color and appearance; however,

212 Ali et al.

Int. J. Biosci. 2015

there was an overall dominance of wild traits in these

hybrids. Dominance of wild traits were apparent in

panicle characters such as panicle type, panicle

exertion and grain characters such as grain awning

and grain shattering etc. The hybrid plants were

highly vigorous and showed high tillering ability (Fig.

1). Degree of cross affinity varied with the genetic

makeup of female parents (Table 1), suggesting that

different cultivars have different cross affinity with

their ancestral wild species. Among the

morphological markers, the anthocyanin

pigmentation proved to be a very useful one. At a

very early stage (3 to 4 days after germination), the

hybrid nature of F1 seedlings were confirmed through

the basal leaf sheath coloration, as the female parents

lack the pigment (anthocyanin) in their basal leaf

sheaths whereas the basal leaf sheath of hybrids and

male parent were pigmented.

Table 2. Meiotic configuration of IR-6, O. rufipogon (Acc. 106516) and their F1 hybrids.

Chromosome pairing IR-6 O. rufipogon IR-6 x O. rufipogon

DK M1 DK M1 DK M1

Cell analyzed(no) 42 26 94 52 46 100

Univalents

Range

Mean/cell

0 – 2

0.10

0 – 2

0.08

0 – 2

0.21

0 – 2

0.14

0 – 8

1.61

0 – 4

0.37

Total Bivalent

Range

Mean/cell

11 – 12

11.95

11 – 12

11.96

11 – 12

11.75

10 – 12

11.90

8 – 12

11.20

8 – 12

11.62

Rings

Range

Mean/cell

10 – 12

11.88

11 – 12

11.96

10 – 12

11.69

10 – 12

11.64

8 – 12

10.83

5 – 12

11.02

Rods

Range

Mean/cell

0 – 2

0.07

_

_

0 – 2

0.07

0 – 2

0.27

0 – 4

0.37

0 – 6

0.60

Trivalent

Range

Mean/cell

_

_

_

_

_

_

0 – 1

0.02

_

_

0 – 1

0.05

Quadrivalents

Range

Mean/cell

_

_

_

_

_

_

_

_

_

_

0 – 2

0.06

Chiasma/ PMC

Range

Mean/cell

22 – 24

23.86

22 – 24

23.92

22 – 24

23.46

22 – 24

23.58

16 – 24

21.50

16 – 24

22.98

DK=Diakinesis M1=Metaphase-I

Meiotic configuration was recorded as range and mean per cell.

Variations in awn color were observed at early

flowering. O. sativa possessed white color awns while

O. rufipogon manifested red color awns. The F1

hybrids showed red & white color awns. The upper

portion of awn was red while the lower portion was

white (Fig. 2).

Cytogenetic characterization of O. sativa, O.

rufipogon and their F1 hybrids

Meiotic chromosome pairing of O. sativa (IR-6, KS-

213 Ali et al.

Int. J. Biosci. 2015

282 and Bas-385), O. rufipogon and their F1 hybrids

were analyzed at diakinesis and metaphase-I. From

the cytological observations, it was confirmed that the

parental species and their F1 hybrids, had a consistent

chromosome number of 2n=2x=24 in meiotic PMCs

with normal meiosis. The analysis of meiosis showed

that chromosome pairing of IR-6, Bas-385 and KS-

282 was normal at diakinesis/metaphase-I. No

trivalents or quadrivalents were observed. However,

univalents were observed at a very low frequency. The

mean chromosome configuration of IR-6 at diakinesis

was 0.10 I + 11.95 II (11.88 rings + 0.07 rods), with

23.86 chiasma per cell, while it was 0.08 I + 11.96 II,

with 23.92 chiasma per cell, at metaphase-I.

Table 3. Meiotic configuration of KS-282, O. rufipogon (Acc. 106516) and their F1 hybrids.

Chromosome pairing KS-282 O. rufipogon KS-282 x O. rufipogon

DK M1 DK M1 DK M1

Cell analyzed(no) 53 33 94 52 109 49

Univalents

Range

Mean/cell

0 – 2

0.15

0 – 2

0.18

0 – 2

0.21

0 – 2

0.14

0 – 12

0.92

0 – 22

1.20

Total Bivalent

Range

Mean/cell

11 – 12

11.92

11 – 12

11.91

11 – 12

11.75

10 – 12

11.90

6 – 12

10.36

1 – 12

10.96

Rings

Range

Mean/cell

9 – 12

11.81

10 – 12

11.82

10 – 12

11.69

10 – 12

11.64

5 – 12

9.68

1 – 12

9.96

Rods

Range

Mean/cell

0 – 3

0.11

0 – 2

0.09

0 – 2

0.07

0 – 2

0.27

0 – 6

0.67

0 – 4

1.00

Trivalent

Range

Mean/cell

_

_

_

_

_

_

0 – 1

0.02

0 – 2

0.06

0 – 1

0.02

Quadrivalents

Range

Mean/cell

_

_

_

_

_

_

_

_

0 – 1

0.02

0 – 2

0.20

Chiasma/ PMC

Range

Mean/cell

21 – 24

23.74

22 – 24

23.73

22 – 24

23.46

22 – 24

23.58

12 – 24

20.21

2 – 24

21.78

Meiotic configuration was recorded as range and mean per cell.

Mean chromosome configuration of KS-282 at

diakinesis was 0.15 I + 11.92 II (11.81 rings + 0.11

rods), with 23.74 chiasma per cell, whereas it was

0.18 I + 11.91 II (11.82 rings + 0.09 rods), with 23.73

chiasma per cell, at metaphase-I. Similarly, mean

chromosome configuration of Bas-385 at diakinesis

was 0.14 I + 11.93 II (11.89 rings + 0.04 rods), with

23.82 chiasma per cell and it was 0.05 I + 11.97 II

(11.87 rings + 0.10 rods), with 23.85 chiasma per cell,

at metaphase-1.

Mean chromosome configuration of O. rufipogon was

0.21 I + 11.75 II (11.69 rings + 0.07 rods), with 23.46

chiasma per cell at diakinesis, whereas it was 0.14 I +

11.90 II (11.64 rings + 0.27 rods) + 0.02 III, with

23.58 chiasma per cell, at metaphase-I. Thus,

chromosome configuration did not differ considerably

214 Ali et al.

Int. J. Biosci. 2015

at diakinesis and metaphase-I. However, trivalent

were observed only at metaphase-I of O. rufipogon

and were lacking at diakinesis. Chromosomes were

equally segregated to the two poles at anaphase-I and

–II. Thus, both O. sativa and O. rufipogon showed

normal meiosis.The F1 hybrids also showed regular

meiosis (Table 2, 3 and 4).

Chromosome configuration at diakinesis/metaphase-

I of all the F1 hybrids did not differ appreciably from

those of their parental species. However, the

frequencies of univalents and rod bivalents were quite

high as compared to the parental species. Trivalents

and Quadrivalents were observed only at metaphase-

I. Chiasma frequency was relatively low as compared

to their respective parents. This reduction in chiasma

frequency was due to the high occurrence of

univalents and rod bivalents in these hybrids. Mean

chromosome configuration of IR-6 × O. rufipogon at

diakinesis was 1.61 I + 11.20 II (10.83 rings + 0.37

rods), with 21.50 chiasma per cell. While it was 0.37 I

+ 11.62 II (11.02 rings + 0.60 rods) + 0.05 III + 0.06

IV, with 22.98 chiasma per cell, at metaphase-I.

Table 4. Meiotic configuration of Bas-385, O. rufipogon (Acc. 106516) and their F1 hybrids.

Chromosome pairing Bas-385 O. rufipogon Bas-385x O. rufipogon

DK M1 DK M1 DK M1

Cell analyzed(No) 100 39 94 52 57 36

Univalents

Range

Mean/cell

0 – 4

0.14

0 – 2

0.05

0 – 2

0.21

0 – 2

0.14

0 – 8

1.04

0 – 4

0.47

Total Bivalent

Range

Mean/cell

10 – 12

11.93

11 – 12

11.97

11 – 12

11.75

10 – 12

11.90

8 – 12

11.40

10 – 12

11.69

Rings

Range

Mean/cell

10 – 12

11.89

11 – 12

11.87

10 – 12

11.69

10 – 12

11.64

8 – 12

10.95

9 – 12

11.44

Rods

Range

Mean/cell

0 – 1

0.04

0 – 1

0.10

0 – 2

0.07

0 – 2

0.27

0 – 2

0.46

0 – 3

0.25

Trivalent

Range

Mean/cell

_

_

_

_

_

_

0 – 1

0.02

0 – 1

0.02

0 – 1

0.03

Quadrivalents

Range

Mean/cell

_

_

_

_

_

_

_

_

0 – 10

0.02

0 – 1

0.03

Chiasma/PMC

Range

Mean/cell

20 – 24

23.82

22 – 24

23.85

22 – 24

23.46

22 – 24

23.58

16 – 24

22.44

20 – 24

23.33

Meiotic configuration was recorded as range and mean per cell.

Mean chromosome configuration of KS-282 × O.

rufipogon at diakinesis was 0.92 I + 10.36 II (9.68

rings + 0.67 rods) +0.06III + 0.02 IV, with 20.21

chiasma per cell. On the other hand, it was 1.2 I +

10.96 II (9.96 rings + 1.0 rods) + 0.02 III + 0.2 IV,

with 21.78 chiasma per cell, at metaphase-I. Mean

chromosome configuration of Bas-385 × O. rufipogon

at diakinesis was 1.04I + 11.40 II (10.95 rings + 0.46

rods) +0.02III + 0.02 IV, with 22.44 chiasma per cell.

Whereas at metaphase-I, it was 0.47 I + 11.69 II

(11.44 rings + 0.25 rods) + 0.03 III + 0.3 IV, with

23.33 chiasma per cell.

Chromosomes were equally segregated to the two

215 Ali et al.

Int. J. Biosci. 2015

poles at anaphase-I and –II. Most of the cells showed

normal meiosis. However, straggling chromosomes at

metaphase-I (Fig. 3) and laggards at anaphase-I were

commonly observed. The high frequency of laggards

at anaphase-I was obviously as a consequence of high

frequency of univalents at diakinesis/metaphase-I in

the F1 hybrids. No micronuclei were observed at

telophase-I or –II. However, few chromosome bridges

were observed at anaphase-I. A very few cells with

abnormal spindle orientation, early separation and

late disjunction were also observed (Table 5 and Fig.

3).

Table 5. Chromosome behavior at anaphase-I and telophase-I in parents and their hybrids.

Parent/hybrids Cells analyzed (No) Percent (%) cells observed

Normal Laggards Bridges Bridges + Fragments Abnormal spindle Late disjunction

O. rufipogon 120 96.6 1.7 1.7 - - -

IR-6 186 97.85 2.15 - - - -

KS-282 198 98.98 1.01 - - - -

BAS-385 103 98.06 1.94 - - - -

IR-6x O.rufipogon 136 87.51 4.41 1.47 - 2.94 2.2

KS-282x O.rufipogon 190 78.90 8.42 0.53 1.05 6.32 3.2

Bas-385x O.rufipogon 175 86.26 5.14 0.57 - 3.43 4.6

Discussion

In the genus Oryza, interspecific hybrids are useful

bridges for transferring the desired genes from wild

species to cultivated rice (O. sativa L.). In this study

interspecific crosses were made between cultivated

varieties and wild species of rice (O. rufipogon Griff.).

Incrossability barriers such as hindrance in pollen

germination, embryo abortion, low cross ability and

poor pollen fertility were observed during the present

study. Gibberellic acid (75 mg/liter) was used to

induce pollen germination and a mixture of 100

mg/liter gibberellic acid (GA3) and 25 mg/liter

naphthalene acetic acid (NAA) was used to prevent

embryo abortion.

Fig. 1. Morphology of interspecific hybrid (KS-282 ×

O. rufipogon),that shows purplish green color basal

leaf sheaths (arrow), high tillering ability and

spreading growth habit.

The cross ability between O. sativa and O. rufipogon

was very low (< 10 %) and varied from 4.35-8.33 (%).

The highest cross ability was observed in IR-6 × O.

rufipogon (8.33%) while the least one in KS-282 × O.

rufipogon (4.35%). Based on cross ability, IR-6

showed a close affinity with O. rufipogon. Our

findings indicate that cross ability between O. sativa

and O. rufipogon varied with the genetic makeup of

the female parents. Thus different cultivars have

different cross affinities with their ancestral wild

species. Similar variations in cross ability have been

reported by many researchers e.g. Guo et al., 2009

and Naredo et al., 1998.

Fig. 2. Variation in awn color of parents and their

hybrids (a) red color awn of O. rufipogon, (b) red &

white color awn of F1 hybrid, “the upper half portion

is red (small arrow) while lower half portion is white

(large arrow)”, (c) white color awn of O. sativa cv.

Bas-385.

216 Ali et al.

Int. J. Biosci. 2015

All the hybrid plants obtained from the cross of O.

sativa × O. rufipogon were intermediate between two

parents in characters such as basal leaf sheath color

but wild traits like open type panicles, high grain

shattering and grain awning dominated in the

hybrids. Regarding the dominance of wild traits,

similar observations were reported in several

intragenomic and intergenomic hybrids of rice (Brar

et al., 1991; Naredo et al., 1998; Niroula et al.,

2009). Several approaches of plant breeding

demand constant reference to the chromosomal

status of the breeding materials. The degree of

variability, viability and stability desired in the

plant breeding often depends on the efficiency of

parental chromosomes pairing and subsequent

recombination. In order to assess genomic affinity

between cultivars of O. sativa and O. rufipogon,

chromosome behaviors in pollen mother cells

(PMCs) of parents and their hybrids were

analyzed at diakinesis, metaphase-I, anaphase-I

and telophase-I. The frequency of abnormalities

such as univalents (I), trivalents (III) and

quadrivalents (IV) were slightly high in all hybrids.

Despite the low occurrence of such abnormalities,

on an average all the interspecific hybrids showed

normal meiosis with remarkably high degree of

chromosome pairing. At metaphase-I, all the

hybrids had more than 10.96 bivalents and 21.78

chiasma/PMC.

Fig 3. Meiotic behavior of interspecific hybrid (O. sativa cv. Bas-385 × O. rufipogon). (a) diakinesis showing 12

bivalents (11 rings + 1 rod), (b) Metaphase-I showing two straggling bivalents (arrow), (c) Cytokinesis with

abnormal spindle orientations (arrow), (d) Tetrad with abnormal nuclei (arrows).

Meiotic behavior in this study was regular in all the

interspecific hybrids at metaphase I. Chromosome

configurations of the interspecific hybrids were not

differed appreciably from those of the parental

species. The total number of bivalents ranged from

10.36-11.69/PMC with majority of ring bivalents.

Chiasma frequencies were also comparable with

parental species and varied from 20.21 to 23.33/cell.

Chromosomes were equally segregated to the two

poles at anaphase- I and II in most of the PMCs.

However a very few cells with early separation, late

disjunction and abnormal spindle orientation were

recorded at anaphase-I in the F1 hybrids. Based on

meiosis of intervarietal and interspecific hybrids,

Shastry (1966) suggested that AA genome species are

differentiated by chromosome structural changes to

various degrees. However in this study, the

chromosome pairing in all F1 hybrids at metaphase-I

was essentially normal. The type and frequency of

meiotic aberrations observed in this study are similar

217 Ali et al.

Int. J. Biosci. 2015

to those found in interracial hybrids in O. sativa

(Nayar, 1973; Kumaran and Menon, 1982).

Univalents and quadrivalents were

frequentlyobserved not only in interspecific

hybrids, but also commonly reported for

interracial and intervarietal hybrids and even in

true parental forms (Nayar, 1973; Kumaran and

Menon, 1982).

Although the range of univalents was quite high in

this study (0-22) in KS-282 × O. rufipogon, the

average number of quadrivalents and univalents

were similar to those reported earlier. Similar

observations were also reported in cross involving

O. sativa and O. nivara (Niroula et al., 2009).

High frequency abnormalities such as frequent

formation of 0-3 quadrivalents, univalents and

chromosomal elimination have been reported in

O. sativa × O. rufipogon hybrids earlier

(Majumder et al., 1997). Occurrence of structural

changes at later stages such as quadrivalents

formation at diakinesis and metaphase-I and

bridges and fragments at anaphase-I can be taken

as an indicator of translocation and inversion,

respectively. However, the low frequency of such

anomalies observed in this study is not enough to

generalize the structural differentiations. Bridges

and fragments were also observed in 11 of the

interracial hybrids out of 33 and it was concluded

that bridges and fragments mostly occurred by

inversion (Nayar, 1973). Several other

investigators reported occurrence of anaphase

bridges in interspecific crosses without mentioning

their frequencies (Kumaran and Menon, 1982;

Ishii et al., 1995). High occurrence of bridges

without fragments at anaphase is mostly

attributed to delay terminalization of chiasmata,

sticky bivalent formation, or breakage and random

reunion of the chromatids at earlier stages

regardless of homology as reported by Walter

(1963).

The results obtained herein clearly indicated that

the failure of bivalent formation at the later stages

in hybrids is not always a proof of lack of

homology. Such failure of synapsis can possibly be

brought by many external and internal factors as

reported by Misra and Shastry (1969). The present

data thus indicates that O. rufipogon and O. sativa

has most likely the same genomic composition.

Conclusions

Common wild rice O. rufipogon produced viable

F1 hybrids when crossed with three widely grown

cultivated varieties of Pakistan viz. Bas-385, IR-6

and KS-282. The use of growth regulators GA3

and NAA proved to be very useful triggering

agents in making interspecific crosses. F1 hybrids

were confirmed easily through morphological

markers. The F1 hybrids showed normal meiosis

with remarkably high degree of chromosome

pairing at metaphase-I. Therefore, favorable

alleles of resistant genes can be easily

introgressed from O. rufipogon into cultivated

rice.

Acknowledgements

We highly acknowledge Higher Education

Commission (HEC) Pakistan for funding this

research project under PhD fellowship program.

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