Improving Productivity and Livelihood for Fragile Environments

8/9/2019 Improving Productivity and Livelihood for Fragile Environments

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Improving productivity

and livelihood for fragile

environments

T B 2008 No. 13


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The International Rice Research Institute (IRRI) was established in 1960

by the Ford and Rockefeller Foundations with the help and approval of

the Government of the Philippines. Today, IRRI is one of the 15 nonprot

international research centers supported by the Consultative Group on In-

ternational Agricultural Research (CGIAR www.cgiar.org).

IRRI receives support from several CGIAR members, including theWorld Bank, European Union, Asian Development Bank, International

Fund for Agricultural Development, Rockefeller Foundation, Food and

Agriculture Organization of the United Nations, and agencies of the fol-

lowing countries: Australia, Brazil, Canada, Denmark, France, Germany,

India, Iran, Japan, Malaysia, Norway, Peoples Republic of China, Repub-

lic of Korea, Republic of the Philippines, Sweden, Switzerland, Thailand,

United Kingdom, United States, and Vietnam.

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Rice Research Institute.

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(2008) and is licensed for use under a Creative Commons Attribution-Non-Commercial-ShareAlike 3.0 License (Unported). Unless otherwise noted, users arefree to copy, duplicate, or reproduce, and distribute, display, or transmit any of thearticles or portions of the articles, and to make translations, adaptations, or otherderivative works under the following conditions:

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Phone: +63 (2) 580-5600

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Suggested Citation:

IRRI (International Rice Research Institute). 2008.

Improving productivity and livelihood for fragile environments.IRRI Technical Bulletin No. 13. Los Baos (Philippines): IRRI. 54 p.

Editing: Bill Hardy

Layout and Design: Emmanuel Panisales

ISSN 0074-7807


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Contents

Contents....................................................................................................................III

Preface......................................................................................................................IV

Physiological basis of tolerance of ash ooding during germination

and early seedling establishment in rice..........................................................................1

Abdelbagi M. smail, Evangelina S. Ella, Gina Vergara, Donna F. Holt-Stevens,

Alvaro Pamplona, and Dave Mackill

Salinity tolerance in rice: physiological bases and implications

for management strategies for better crop establishment.................................................8

Abdelbagi M. smail, Babita Thapa, and James Egdane

Opportunities for direct seeding and improved weed control in the

Barind of Bangladesh...................................................................................................15

M.A. Mazid, C.R. Riches, A.M. Mortimer, and D.E. Johnson

Breeding for submergence tolerance.............................................................................20D.J. Mackill, A.M. smail, S. Heuer, E. Septiningsih, A.M. Pamplona, R.M. Rodriguez,C.N. Neeraja, D. Sanchez, K. ftekhar, and G. Vergara

Participatory varietal selection of salinity-tolerant rice for the

coastal wetlands of Bangladesh....................................................................................27

M.A. Salam, G.B. Gregorio, D.L. Adorada, and R.D. Mendoza

Increasing prots from rice production in Bangladesh:

direct wet seeding of rice using a plastic drum seeder.....................................................32M. Zainul Abedin

Characterizing and understanding the socioeconomic conditions

of farming households in rainfed rice environments: a case

in eastern Uttar Pradesh..............................................................................................36

T. Paris, A.D. Cueno, and A. Singh


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V

Preface

Improving Productivity and Livelihood for Fragile Environments is one of

IRRIs four programs and 11 projects stipulated in its Medium-Term Plan

for 2006-2008. The Medium-Term Plan (MTP) for 2006-2008 reects IRRIs

core agenda in addressing current and emerging problems in rice. It is guided

by the broad framework of the strategic plan outlined in the document

IRRI Toward 2020 published in 1996, and updated in November 2003. It also

takes advantage of the scientic opportunities that assist the Institute in

reaching its goals.

Improving Productivity and Livelihood for Fragile Environments is

Program 3 of the MTP for 2006-2008. It examines risk reduction in rice

cultivation. The program also focuses on helping increase yield and farm

income via the development of stress-tolerant beer-yielding varieties

using ecient crop management practices.

In the past, the probability of success in research for building tolerance for

abiotic stresses into beer-yielding varieties was low, leading to inadequate

allocation of research resources to solve these problems. But with the

recent advances in molecular biology for tagging and characterizing

genes and their transfer to other species, the probability of success in this

area brightened. Since the environments are diverse and their domains

vary across countries, the research is being done in partnership with thenational agricultural research and extension systems, drawing on local

scientic expertise and farmers indigenous knowledge.

Program 3: Improving Productivity and Livelihood for Fragile

Environments aempts to solve the abovementioned problems in three

projectsProject 7: Genetic enhancement for improving productivity and human

health in fragile environments; Project 8: Natural resource management for rainfed

lowland and upland rice ecosystems; andProject 9: Consortium for Unfavorable

Rice Environments (CURE).

This Technical Bulletin showcases the studies and research results fromeach of the projects under Program 3.


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More than 16 million ha of lowland and

deepwater rice areas are unfavorably aected

by ooding because of complete submergence.

Rice is the only crop plant adapted to aquatic

environments because of its well-developed

aerenchyma tissue that facilitates oxygen

diusion through continuous air spaces from

shoot to root and avoids anoxia development

in roots. However, complete submergence

due to frequent ooding can adversely aect

plant growth and yield. Two types of ooding

cause damage to rice: ash ooding, which

results in rapid ascending of water levels with

complete submergence for about 12 weeks,

and deep water, in which water depth exceeds

100 cm and persists for longer periods of up to

several months. Plants may become completely

submerged for short periods if ooding

is severe. Elongation ability of leaves and

internodes is essential in deepwater conditionsto keep pace with the rising water levels and

to escape complete submergence. Traditional

varieties adapted to these environments are

low yielding because of their low-tillering

ability, susceptibility to lodging, and poor grain

quality.

Flash ooding can occur any time during

the growing season and usually occurs more

than once. It is usually more damaging if

it occurs early in the season, particularly

during early crop establishment. This isbecause younger seedlings are more sensitive

to ooding. In direct-seeded areas, even

waterlogging can be devastating because of the

high sensitivity of all crop plants to low oxygen

during germination.

Physiological basis of tolerance of ash ooding duringgermination and early seedling establishment in rice

Abdelbagi M. smail, Evangelina S. Ella, Gina Vergara, Donna F. Holt-Stevens, Alvaro Pamplona, and Dave Mackill

Tolerance of ooding during germinationand early seedling establishment

The likelihood of occurrence of ash ooding

at dierent stages of growth requires dierent

tolerance strategies. In rainfed lowlands, direct

seeding is becoming more popular because of

the escalating expense and scarcity of labor, and

farmers are also enthusiastic to adopt it because

of its additional benets such as shorteningcrop duration and the enhanced tolerance of

some stresses, particularly drought. Breeding

cultivars with tolerance of ooding during

germination and early seedling establishment

will help avoid crop failures commonly

encountered when early ooding occurs in both

rainfed and irrigated ecosystems. Moreover,

maintaining a shallow water head aer

seeding under irrigated conditions can help

suppress weed growth and this could provide

an eective, cheap, and sustainable methodfor weed management. However, this practice

is stalled by the unavailability of suitable

germplasm. Previous studies showed that rice

is capable of germination underwater, but this

capability is limited to coleoptile elongation,

with failure to develop further.

In an aempt to discern rice germplasm

with higher tolerance of submergence during

germination, we screened more than 8,000

genebank accessions and breeding lines and

a few tolerant lines were identied. Screeningwas conducted by direct dry seeding in plastic

trays containing a shallow layer of soil. Seeds

of individual lines were sown in rows and

immediately submerged by adding water to a

depth of 10 cm. This depth is maintained for

23 weeks, when the percentage of seeds that

germinate and successfully emerge from water


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is determined. Through this method, about 12

lines were identied as being tolerant (Table 1)

and the tolerance of some of them was further

conrmed in replicated trials (Fig. 1A,B).

Traits associated with tolerance of oodingduring germination and early growth

Tolerant lines and a few intolerant checks were

evaluated for some agronomic traits to test

their association with tolerance. A subset of

these is shown in Table 2. Tolerant cultivars

vary substantially with respect to mature plant

height, days to owering, and average grain

weight. This variation provides opportunities

for breeders to select appropriate parental lines

for crossing to incorporate this trait. Tolerant

lines seem to emerge faster from the soil andwater (by about 23 d and 24 d, respectively)

when compared with intolerant lines. The

strong negative correlation of survival with

time of emergence from soil (R = 0.97) and

water (R = 0.95) indicated that fast germination

and growth under hypoxic conditions is crucial

for survival. However, survival did not correlate

with plant height at maturity, days to owering,

or average grain weight.

Tolerant cultivars tend to grow faster

underwater, produce taller seedlings withmore leaves, and aain greater leaf area than

intolerant lines (Fig. 2), traits

that might also be useful for

weed competitiveness early

in the season.

Germination under anoxia(absence of O2)

Six tolerant and two sensitive

cultivars were evaluated for

their ability to germinateunder anoxia. Seeds were

sterilized using sodium

hypochloride and tween

20, and then placed in a

1% stagnant agar solution

deoxygenated by continuous

bubbling of nitrogen gas.

Table 1. Rice accessions most tolerant of ooding during

germination.

Cultivar Origin Percent survival

(rst screening)

Khaiyan 90

Khao Hlan On Myanmar 75

Cody United States 70

Dholamon 64-3 Bangladesh 80

Liu-Tiao-Nuo China 70

Ma-Zhan (Red) China 90

Sossoka Guinea 85

Kaolack Guinea 85

Kalonchi 90

Nanhi ndia 80

R6855-00---- NPT-RR 75

R6855-00--3- NPT-RR 55

Fig. 1. Average survival of selected lines over two separate replicated experiments. Vertical bars

indicate (A) S.E. and (B) LSD0.05.

00

80

60

40

0

0Khaiyan Khao Hlan On FR3A R4

Tolerant ntolerant

Seedling survival (%)

Not submergedSubmerged

60

80

40

0

0

00

KhaiyanCody Kalonch Nanh Khao

Hlan On

FR3A R R8 R4 R64

Tolerant ntolerant

Seedling survival (%) after dr y seeding and submergence for 21 d

LSD0.05

A

B


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Evaluation of percent germination, length of

coleoptiles, as well as appearance of roots was

carried out aer 7 d of continuous anoxia.

Tolerant lines had greater coleoptile length

under both anoxic and normal conditions

(Fig. 3).

Physiological mechanisms associated withtolerance of ooding during germination

Total amylase activity

The ability to break down starches into simple

sugars is a major factor limiting germination

under ooded conditions. This is because of

the high sensitivity of key enzymes involved

Table 2. Comparison between selected tolerant and sensitive lines for selected agronomic traits and their associa-

tion with survival.

Cultivar Origin Days to owering Mature height

(cm)

1,000-seed wt. Days to emergence Survival (%)

Soil Water

Tolerant lines

Dholamon Bangladesh 7 03 .5 4 9 8

Liu-Tiao-Nuo China 6 43 7.0 4 9 7

Khaiyan ? 66 60 8. 5 74

Khao Hlan On Myanmar 85 45 8.3 4 9 73

Intolerant lines

R64 Philippines 65 30 0.0 7 3 7

FR3A ndia 70 67 9.7 7 3 0

R4 Philippines 73 5 8.4 7 3 9

Correlation with

survival

0.47 0.50 0.00 0.97 0.95

8

6

4

0Seedling height (cm) Root length (cm) Leaf area (cm)

Fig. 2. Average shoot height, root length, and leaf area of ve tolerant

(shaded columns) and three intolerant (black columns) cultivars

germinated underwater. Results are means of 3 replicates taken

21 d after seeding.

in this process to low oxygen. Total amylase

activity was measured using two tolerant and

two sensitive lines germinated in ooded

soils for 3 days. The activity of these enzymes

increased substantially in germinating seeds of

tolerant lines under ooding but did not change

signicantly in sensitive lines (Fig. 4). Higheractivity of these enzymes in germinating seeds

of tolerant lines under ooding indicated that

these lines are able to break down stored starch

into simple sugars beer than sensitive lines,

and this step is essential for germination and

growth. Metabolic breakdown of simple sugars

is much less sensitive to low oxygen than the

Fig. 3. Average coleoptile length of six tolerant (open

columns) and two intolerant (black columns) cultivars under

anoxia and aerated conditions. Data are from 7-d-old seedlings

and vertical bars indicate S.E.

0Aerated tap water Anoxic agar

3Coleoptile length (cm) Coleoptile length (cm)


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processes involved in starch degradation.

Total activities of these enzymes correlated

positively with shoot and root length (R = 0.85,

0.83, respectively), and with plant survival (Fig.

4B), suggesting a strong relationship between

the activity of these enzymes and the ability of

seeds to germinate and seedlings to grow under

submerged conditions.

Plant hormones

Germinating seeds of tolerant lines produced

more ethylene than susceptible ones under

submergence (Fig. 5). Ethylene is known

to counteract ABA synthesis and increase

synthesis of and sensitivity to GA. High

levels of ABA in seeds are associated with the

inhibition of seed germination and dormancy;

however, high GA is associated with activation

of amylase enzymes and breakdown of stored

starches to be used for further growth ofgerminating seeds.

Peroxidase activity

Peroxidases are responsible for the assembly

of lignins and proteins in the cell wall and

for the binding of ferulic acid to cell walls

by the formation of diferuloyl cross-links to

matrix polysaccharides, both of which are

associated with reduced cell wall extensibility.

Higher peroxidase activity is closely associatedwith reduced growth in other plants such

as in mung bean and peanut. We measured

peroxidase activity in germinating seeds of two

tolerant (Khaiyan and Khao Hlan On) and two

sensitive (R64 and IR22) cultivars to evaluate

its involvement in growth under low-oxygen

conditions. Tolerant genotypes showed much

50

40

30

0

0

0Khaiyan Khao Hlan On FR3A R4

Tolerant ntolerant

Not submergedSubmerged

Amylase activity (units mg protein)

0

4

6

0 0 30 40 50Amylase activity

Plant survival (%)

R = 0.9

Khao Hlan On (N)

Khao Hlan On (S)

IR42 (N)

IR42 (S)

30

5

0

5

0

5

0

Ethylene content (mg seedling)

4 5 7 8 0Days of incubation in soil

Fig. 5. Ethylene content of germinating seeds of one tolerant and one

sensitive line under submerged conditions. N = normal, S = stress.

Fig. 4. (A) Total amylase activity in germinating seeds of two tolerant and two intolerant rice cultivars sown in submerged soils for 3 days.

Vertical bars are S.E. (B) Correlation between amylase activity and seedling survival.

A B


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less peroxidase activity than intolerant ones

(Fig. 6A). A strong negative correlation was

observed between peroxidase activity and

shoot growth (R = 0.69) and seedling survival

(Fig. 6B). The ability of seedlings to elongate

underwater and emerge quickly is critical forsurvival. This may also suggest that lower

peroxidase activity in seedlings germinating

under anoxia could be used as an indicator for

ability to germinate and grow under ooded

conditions.

Nonstructural carbohydrate content and ex-

pression of key enzymes involved in carbo-

hydrate catabolism

Nonstructural carbohydrate content. Seed starch

and soluble sugars are the main sources ofenergy for embryo growth in germinating seeds

and, under anaerobic conditions, breakdown of

starch into sugars decreases greatly because of

reduced activity or expression of the enzymes

involved in starch catabolism under low-oxygen

conditions.Comparisons oftotal starch levels

were made between tolerant Khaiyan and

intolerant IR42 from 0 to 5 d of hypoxia (0.03

mol O2 m3). We found that total starch was

always higher for the tolerant cultivar for up to

5 d of hypoxia and did not change signicantlybetween time points. The availability of total

soluble sugars (TSS) was compared between

tolerant Khaiyan and intolerant IR42 at 0

d, 1 d, 3 d, and 5 d of hypoxia. TSS levels at

0 d (25 to 32 g per seed dry wt.) were not

signicantly dierent between tolerant and

intolerant rice cultivars. However, aer 1 d of

hypoxic treatment, availability of TSS declined

rapidly, by 60%, and then increased at 3 d in

germinating embryos. We found signicant

dierences in TSS between tolerant Khaiyanand intolerant IR42 starting at 3 d and up to

5 d of hypoxia (Fig. 7). The increased levels

of available TSS may stem from the higher

carbohydrate breakdown brought about by

the higher amylase activity found in Khaiyan,

which in turn contributes to the greater ability

of Khaiyan to survive ooding and low-oxygen

stress during germination.

Alpha-amylases. Starches are a major energy

source for developing rice embryos and the

total amylases needed to break them down

were generally found to be higher for tolerant

cultivars under low-oxygen or submergedconditions than for intolerant lines. Analyses

of transcriptional activity of rice -amylases

(RAmy1A, RAmy2A, RAmy3C, RAmy3D, and

RAmy3E) were performed using RT-PCR of

RNA extracted from germinating embryos

of tolerant (Khaiyan) and intolerant (IR42)

genotypes subjected to hypoxia: air (0.25 mol O2

m3) or anoxia (


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0 h, 12 h, 24 h, 48 h, and 72 h. Rice -amylases

(RAmy1A, RAmy3C, RAmy3D, and RAmy3E)

generally showed up-regulation starting at 12

h following treatment (Fig. 8). RAmy2A was not

expressed. During hypoxia, RAmy3D, which

is important for oligosacharride degradation,

showed relatively higher expression in the

tolerant Khaiyan than in the intolerant IR42.

RAmy1A, which breaks down soluble starch,

was less induced during anoxia but not during

hypoxia in tolerant cultivars. No dierencesin expression levels were found for RAmy3C

and RAmy3E in the three O2 regimes. Using

RT-PCR analyses, we found that rice -

amylases were rapidly induced in germinating

embryos. RAmy3D, a key and major enzyme for

carbohydrate use for germinating embryos, was

expressed at higher levels in tolerant cultivars

during hypoxia and this probably helps provide

and sustain energy during germination.

Sucrose synthases. Sucrose is another major

energy source for germinating embryos. Itcan be hydrolyzed via the sucrose synthase

(Sus) pathway or the invertase pathway. Under

low-O2 stress, Sus transcript levels increase

while those of invertase decrease, suggesting

that sucrose synthase is the principal enzyme

that converts sucrose to phosphorylated

hexose under low oxygen. Comparison of

Treatment days

0 3 5

35

30

5

0

5

0

5

0

Total soluble sugars (g per seed dry wt.)

Khaiyan

R4

Fig. 7. Total soluble sugars of Khaiyan and IR42 at different time

points after hypoxia treatment.

transcriptional activity of sucrose synthase

paerns in tolerant and intolerant rice embryos

showed no dierences between treatments

under aerated conditions or during hypoxia

or anoxia. We showed the induction of Sus1

during the rst 72 h of treatment, whereas Sus3activity was completely shut down aer 24 h of

treatment (Fig. 8). Overall, the result suggests

that Sus1, but not Sus3, appeared to contribute

to sucrose degradation during germination.

Pyruvate decarboxylases and alcohol

dehydrogenases. Rice seedlings under low-oxygen

stress are capable of survival for a limited time

by shiing to the fermentative pathway to

generate ATP for cellular metabolism. Using

RT-PCR analyses, we showed that Pdc1,Adh1,

andAdh2 transcripts were induced duringthe rst 72 h of rice seedling growth whether

in air or under hypoxic or anoxic conditions

(Fig. 8). Although oxidative phosphorylation

provides major ATP needs for rapid growth

under aerobic conditions, our results showed

that germinating rice embryos do experience

anaerobic conditions and partly use the

fermentative pathway to generate energy.

PDC and ADH activity. For total PDC

and ADH enzyme activity levels, marked

dierences were observed between tolerant andintolerant cultivars under hypoxic compared

with aerobic conditions (Fig. 9). PDC activity

was 1.6x higher immediately aer 12 h of

Hours 0 12 24 48 72 0 12 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72

RAmy1A

RAmy3C

Hypoxia Air Anoxia

Khaiyan Khaiyan KhaiyanIR42 IR42

RAmy3E

RAmy3D

Gapdh

Sus1

Sus3

Pdc1

Adh1

Adh2

IR42

Fig 8. RT-PCR of key enzymes involved in carbohydrate

metabolism.


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greater soluble sugars, particularly a few days

aer imbibition under hypoxia, and maintain

higher activity of the key enzymes involved in

anaerobic respiration (ADH and PDC). Further

studies are ongoing to establish the full array

of physiological and biochemical mechanisms

associated with tolerance as well as to unravel

their genetic bases. Backcross populations were

developed and are being used to map this trait.

Crosses were also made and are being selected

and advanced to incorporate tolerance into

modern varieties, for both irrigated and rainfed

ecosystems.

.

0 h

.0

0.8

0.6

0.4

0.

0.0 h 4 h 48 h 7 h

Air KhaiyanAir R4Hypoxia KhaiHypoxia R4

PDC levels (min mg protein)

0 h h 4 h 48 h 7 h0.0

0.

0.4

0.6

0.8

.0

.

.4

ADH levels (min mg protein)

Fig 9. (A) PDC and (B) ADH activities. Means with the same letter are statistically not different at each time point. The asterisks indicate

signicant differences between genotypes and treatment at particular time point (P


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Salinity tolerance in rice: physiological bases and implicationsfor management strategies for better crop establishment

Abdelbagi M. smail, Babita Thapa, and James Egdane

In most coastal areas, salinity is high in both

soil and water during the dry season, mostly

between December and July, and with the

peak during May-June. Salinity then decreases

progressively with time aer the onset of the

monsoon season. This poses great challenges

for farming during the dry season, when

most of these areas are oen le barren or are

grown to limited short-maturing crops when

freshwater resources are available. For wet-season rice, the main problems are encountered

during crop establishment in June-July, when

salinity is still high at the beginning of the

rainy season. This is particularly important

because rice is highly sensitive to salt stress

during early seedling growth, which is a major

obstacle because of high seedling mortality and

diculty in establishing a sucient crop stand.

Varieties with reasonable genetic tolerance as

well as proper management strategies during

crop establishment are needed to ensure a goodstand to signicantly enhance and stabilize

productivity in these coastal areas.

For transplanted rice, damage to young

seedlings is further provoked by the fact

that more salt is absorbed passively through

the injured roots, coupled with the fact that

seedling growth and uptake of nutrients are

greatly hindered during the rst week aer

transplanting and until the seedlings are well

established. Osmotic stress of the soil solution

together with a massive uptake of salts andlow nutrient uptake will make nutritional

deciencies even higher for some minerals and

toxicities of others, causing higher seedling

mortality and poor stand establishment. Based

on our understanding of the mechanisms

associated with tolerance of salt stress, we

aempt to develop management strategies

that can reduce seedling mortality aer

transplanting and enhance crop establishment.

Another objective is to compare rice cultivars

contrasting in tolerance of salt stress for their

responsiveness to such stress-mitigating

options.

Nursery management for enhanced seed-ling survival

The eects of seedling N status, seedling age,and handling at transplanting on seedling

survival aer transplanting in saline soils

were investigated. Two rice lines were used,

a moderately tolerant variety, IR64, and a

tolerant breeding line, IR651-4B-10-3 (referred

to as IR651 henceforth). The two cultivars were

grown in grid plastic trays, with one seedling

per partition and half of the seedlings supplied

with extra N at planting. Twenty-d-old and 40-

d-old seedlings with and without N treatments

were transplanted either aer washing theirroots or with soil le aached to their root

system (unwashed). The trial was repeated once

with 4 replications each time.

Average standard evaluation system (SES)

scores of 6.4 and 1 were observed for plants

grown under salt stress and normal conditions,

respectively (Table 1). Average scores were

signicantly lower for the tolerant cultivar

(5.1) than for the moderately tolerant cultivar

(7.8), suggesting that IR651 experienced less

salt injury and had beer growth under salt

stress than IR64. Likewise, transplanting older

seedlings and seedlings with soil aached

to their roots resulted in substantially beer

overall growth as evidenced from visual

observations. The articially salinized plot

seems ideal for evaluating plants for salt stress


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tolerance where a drastic reduction (53%) inplant establishment was observed compared

with the normal plot (100%). Using older

seedlings and seedlings transplanted with soil

intact on roots showed survival of 58% and

56% compared with 35% and 39% for younger

seedlings and seedlings with their roots

washed before transplanting, respectively. The

salt-tolerant line IR651 maintained signicantly

higher seedling survival than IR64. These

results showed that survival of seedlings

transplanted in saline soils could be enhancedsubstantially if older seedlings of tolerant

varieties were used, particularly if their roots

were protected during transplanting.

Older seedlings have higher shoot and root

dry weights and green leaf area; they also

maintain higher starch content in the shoot, all

of which are positively associated with survival

Table 1. Standard evaluation system (SES) scores and per-

centage survival of rice seedlings as affected by cultivar, age

of seedlings in the nursery, and seedling handling under salt

stress and normal conditions. Measurements were taken 20

d after transplanting.

Variables SES scores % survival

CultivarR65 5.06 55.9

R64 7.75 37.7

Seedling age

0 days old 7.56 35.4

40 days old 5.0 58.

Seedling handling

Roots washed 6.93 38.6

Roots not washed 5.87 55.6

Salinity

EC 8 dS m 6.40 46.9

Normal .00 00.0

Signicance

Cultivar ***a **

Age *** ***

Seedling handling ** ***

Salinity *** ***

LSD0.05 (others) 0.47 3.3

LSD0.05 for salinity 0.3 7.0

a*,**, *** = signicant at P< 0.05, 0.0, and 0.00, respectively.

aer transplanting (Table 2). High nonstructural

carbohydrates in shoot and root could probably

act as a reserve source of energy at the time oftransplanting to overcome the period of slow

growth until the transplanted seedlings retain

their full capacity for photosynthetic carbon

xation.

The older seedlings were taller and

experienced less reduction in plant height

under salt stress than the younger ones. IR651

had more tillers per plant under both saline and

normal conditions and showed signicantly

lower reduction in tiller number under salinity.

Younger seedlings produced more tillers perplant under normal conditions whereas the

older seedlings produced more tillers under

saline conditions and transplanted younger

seedlings showed a higher percent reduction

in tiller number (61%) than the older seedlings

(41%) under salt stress. Salt stress delayed

maturity by about 11 d, with greater delays in

younger seedlings when roots are washed and

in sensitive cultivars (Table 3).

This study demonstrated that salinity stress

at transplanting can reduce seedling survivalby >50% and further decrease grain yield of

surviving plants by about 37%, mainly because

of a reduction in number of panicles per plant

as determined by the number of surviving

tillers and number of spikelets per panicle.

Grain yield under salt stress also correlated

positively with the number of panicles per plant

Table 2. Correlations of seedling survival with dry weight and

carbohydrate content at transplanting.

Trait R-valuea

Seedling dry weight 0.65**

Green leaf area 0.60*

Root dry weight 0.68**

Shoot dry weight 0.6*

Total carbohydrates 0.6**

Stem starch 0.60*

Leaf starch 0.68**

Shoot starch 0.5*

Root starch 0.40 ns

ans = not signicant; *, ** = signicant at P< 0.05 and 0.0, respectively.


15/59

0

and spikelets per panicle and to a lesser extent

with individual grain weight but not with

fertility. This is probably because salinity stress

declined gradually 30 d aer transplanting

to simulate the actual conditions of coastal

saline areas, and number of panicles as wellas spikelets was determined earlier. Fertility,

on the other hand, is mainly aected by pollen

grain development and pollination, both of

which occur at a time when salinity stress is

low. The negative eects on individual grain

weight are probably because of the eect of

early stress on photosynthetic leaf area and

stored assimilates. Older seedlings had higher

dry maer and starch content that correlated

positively with seedling survival under salt

stress.In summary, seedlings transplanted with

protected roots had higher shoot dry maer,

less salt uptake, and earlier maturity. The

positive consequences of using older seedlings

and root protection are more evident in the

salt-tolerant IR651 than in the moderately

tolerant IR64. This enhanced responsiveness

of the salt-tolerant cultivar to stress-mitigating

amendments suggests that combining salt

tolerance with proper nursery and seedling

handling options can substantially improve

crop establishment and early growth in salt-

aected areas, which can later be reected in

higher grain yield.

Nutrient management to enhance seedlingestablishment under direct seeding

Despite the well-documented benecial eects

of Ca+ in mitigating salt stress in dierent

plant species, contrasting observations were

noted in rice. This is probably because in most

of the studies conducted so far, only one or a

few sensitive lines were used. Salt stress oen

coexists with P deciency because aected soils

are oen either acidic (saline) or alkaline (sodic),

and both conditions promote P xation in forms

that are poorly available for plants. Thus, the

induced P deciency in these soils could further

worsen the detrimental eects of salt stress

with consequent high seedling mortality. Salt

tolerance is also associated with restricted toxic

ion absorption and adequate uptake of essential

inorganic nutrients, such as K+, to overcome the

Table 3. Plant height, tiller number at 20 d after transplanting, and days to maturity as affected by cultivar, seedling

age, and handling under salt stress and normal conditions. Values are means of 4 replications.

tem Plant height (cm) Tiller number Days to maturity

Normal EC 8 % change Normal EC 8 % change Normal EC 8 % change

Cultivar

R659 50.5 39.5 7.8 0.37 5.5 46.77 4.6 0.9 6.3

R64 48. 44. 8. 9.8 3.9 60.04 98.0 4.6 6.6

Age of seedlings

0 days old 4.4 33.4 9.3 0.7 4.4 6.44 05.4 8.8 3.4

40 days old 5.5 43. 6. 9.48 5.58 4.3 07.4 6.9 9.5

Seedling handling

Roots washed 5.7 4.9 6.9 9.30 5.0 46.0 06.9 .8 5.9

Roots not washed 48. 40. 6.7 0.80 5.03 53.4 05.9 3.8 7.9

Salinity 49. 4.0 6.6 0.08 5.03 50.0 06.4 7.9 .7

Signicancea LSD0.05 Signicance LSD0.05 Signicance LSD0.05

Cultivar ns ** 0.49 *** .

Age ** 3.7 * 0.50 ns

Seedling handling ns ns *** .04Salinity *** 3. *** 0.5 ** .06

Cultivar*salinity ns *** 0.69 ** 1.98

ans = not signicant; *, **, *** = signicant at P< 0.05, 0.0, 0.00, respectively.


16/59

nutritional imbalances caused by salinity stress.

Here, we aempt to investigate the eects of

calcium and phosphorus in enhancing tolerance

of salt stress and in reducing seedling mortality.

Furthermore, we used rice cultivars contrasting

in salinity tolerance to test whether dierential

responses to additional Ca2+ and P could be

observed in these genotypes. Two experimentswere conducted using culture solutions; in each

experiment, two levels of P (2 and 10 L L 1)

and three levels of Ca2+ (20, 40, and 60 L L1)

were used under normal or salt stress (12 dS

m1) conditions. Three rice cultivars were used,

one tolerant (IR651), one moderately tolerant

(IR64), and one sensitive (IR29).

The tolerant line had higher leaf area, higher

root and shoot dry weight, and lower SES

scores when evaluated 3 weeks aer the start

of the stress treatment (Table 4). The addition

of higher levels of P and Ca2+ enhanced leaf

area; however, P enhanced shoot growth while

Ca2+ enhanced root growth under salt stress.

Higher levels of both nutrients resulted in beer

tolerance of salt stress across cultivars as shown

by the signicantly lower SES scores.

Increasing P from 2 L L1 to 10 L L1 in

the nutrient solution resulted in about a 145%

increase in P concentration in shoots. It also

enhanced the uptake of Mg2+ and Ca2+ andsignicantly reduced Na+ concentration in

plant tissue under salt stress. However, the

eect of higher P was greater in IR64, followed

by IR651, and with IR29 showing an opposite

eect, in which Na+ concentration in plant

tissue increased at higher P. This result suggests

that tolerant and moderately tolerant cultivars

are more responsive to higher P in a nutrient

medium.

Increasing calcium concentration in the

culture solution signicantly reduced sodium

concentration in shoots of all three cultivars

and with a relatively greater reduction

Table 4. Leaf area (cm2 seedling1), shoot and root dry weights (g), and SES scores of rice cultivars

under normal conditions and salt stress (12 dS m1) measured 21 d after the start of a salt-stress

treatment.

tem Leaf area Shoot dry wt. Root dry wt. SES scores

Normal Stress Normal Stress Normal Stress Normal Stress

Cultivar

R9 43.6 9.8 0.407 0.5 0.46 0.054 .00 8.83

R64 54.7 33. 0.460 0.3 0.36 0.079 .9 7.83R65 6.7 36.4 0.444 0.348 0.30 0.03 .9 6.36

Phosphorus

L L 50.6 6.3 0.408 0.70 0.65 0.08 .90 7.83

0 L L 56.8 33.3 0.466 0.33 0.60 0.077 .00 7.5

Calcium

0 L L 5. 4.9 0.45 0.83 0.4 0.066 .00 8.4

40 L L 53.4 7.7 0.454 0.94 0.6 0.08 .9 7.78

60 L L 55.5 36.7 0.444 0.97 0.80 0.089 .9 7.

Mean 53.7 9.8 0.437 0.9 0.37 0.079 .94 7.67

Signifcancea

Salinity (S)

Cultivar (C) *** *** *** ***

Phosphorus (P) *** *** *** ***

Calcium (Ca) *** *** ns ***

** ns *** ***

LSD0.05

Salinity 4. 0.056 0.00 0.5

Cultivar 3.6 0.048 0.08 0.46

P .9 0.039 0.

Ca 3.6 0.08 0.46


17/59

8

6

4

3

0

70

60

50

40

30

0

0

0

K+ total uptake in roots (g SDW) K+ uptake in shoots (g SDW)

.5

R9 R64 R65

3.5

3.0

.5

.0

.5

.0

0.5

0.0R9 R64 R65

.0

.5

.0

0.5

0.0

N+

/K+

ratio in roots N+

/K+

ratio in shoots

Cultivar Cultivar

8

4

3

0

8

6

4

0

0

0

0

30

40

50

60

70

Na+ total uptake in roots (g SDW) Na+ uptake in shoots (g SDW)

Ca 0 ppm

Ca 40 ppm

Ca 60 ppm

Fig. 1. Uptake of Na+ and K+ and Na+/K+ ratio in roots (A, B, C) and shoots (D, E, F) of three contrasting rice cultivars as affected by different

levels of Ca2+ in nutrient solution. SDW = shoot dry weight, RDW = root dry weight. Vertical bars indicate standard error.

A

B

C

D

E

F


18/59

3

with increasing genetic tolerance. Sodium

concentration decreased by 14%, 24%, and 35%

in IR29, IR64, and IR651, respectively, with

increasing Ca2+ concentration from 20 to 60

L L1. This also suggests that the response

to higher calcium under salt stress is strongly

dependent on the level of tolerance, with salt-

tolerant cultivars being substantially moreresponsive. This is also clearly reected in the

total uptake of sodium in the shoot of salt-

tolerant cultivars, in which total uptake was

substantially lower in IR651 under higher

calcium in the nutrient solution (Fig. 1D).

Potassium concentration in plant tissue as well

as total uptake into shoots (Fig. 1E) increased

with increasing calcium concentration.

Consequently, Na+/K+ ratio in shoots decreased

signicantly with increasing concentrations of

calcium in the growth medium (Fig. 1F). Under

salt stress, increasing Ca2+ levels also increased

Ca2+ concentrations in all cultivars, but with a

greater increase in IR651 and IR64, in which it

increased by about 19% and 17%, respectively,

compared with only 8% in IR29. Higher Ca2+

concentrations also reduced Na+/Ca2+ ratios in

all cultivars.

Root total Na+ uptake increased with

increasing Ca2+ concentration in the culture

solution in all cultivars (Fig. 1A), which

contrasts with the trend observed for total

sodium uptake into shoots, particularly in IR651

(Fig. 1D). Salt-tolerant cultivar IR651 showed

the highest total sodium uptake into roots,

whereas salt-sensitive cultivar IR29 showed

the lowest Na+ uptake into roots. This greater

compartmentation of sodium into roots of IR651

may partially explain its greater tolerance of

salt stress, which seems to be further enhanced

by supplementary Ca2+. Higher Na+ in roots

could also act as an osmoticum to allow water

uptake into roots of tolerant lines without

much detriment to shoot growth. Total uptake

of K+ into roots also increased with increasing

calcium concentration in the nutrient solution

and with the uptake being relatively higher

in IR651, followed by IR64. Thus, Na+/K+ ratio

in roots was highest at lower Ca2+ in IR64 and

IR651.

The accumulation of Na+ in rice tissues

under salt stress seems to inuence the overall

nutrient balance by changing the internal ion

concentrations in shoots. Na+ concentration in

plant tissue was negatively associated with K+

(R = 0.61**), Ca2+ (R = 0.30**), and Mg2+ (R =

0.28**), but positively correlated with Na+/K+

ratio (R = 0.89**) under salt stress, suggestingthat the optimum balance of these essential

nutrients could be deleteriously aected with

increasing sodium uptake. Phosphorus showed

a positive correlation with Mg2+ under both

normal and salt-stress conditions. However,

Ca2+ concentration in shoots under salt stress

showed a strong positive correlation with Mg2+

(R = 0.72**), P (R = 0.38**), and K+ (R = 0.29**),

and a negative correlation with Na+. Based

on these ndings, it seems that an addition

of more calcium under saline conditions is

benecial because it helps reduce Na+ uptake

while enhancing the uptake of other essential

nutrients such as magnesium, phosphorus,

and potassium, whose uptake was negatively

aected under higher salt stress.

The synergistic eects of the use of genetic

tolerance combined with the mitigating eects

of P and Ca+ are further summarized in Figure

2, in which a lower ratio of Na+/K+ in the shoot

is taken as an indicator of lower salt injury. The

ratio is very high in IR29 but decreased with the

addition of Ca2+ as well as when both Ca2+ and

P were combined. IR64 with its intermediate

level of tolerance showed a beer response to

both P and Ca2+ when applied separately, and a

.0

.6

.

0.8

0.4

0.0

R9 R64 R65

Cultivar

Cultivar (C)

C + PC + CaC + P + Ca

Na+/K+ ratio in shoots

Fig. 2. Na+/K+ ratio in shoots as affected by genotype and P and Ca2+

in three rice cultivars contrasting in their response to salt stress.


19/59

4

greater response when the two nutrients were

combined. IR651, on the other hand, showed

a response similar to that of IR29 but with a

strong genetic eect, particularly when the two

nutrients were added together. A genotype

eect is also obvious. Na+/K+ ratio in plant

tissue drops to


20/59

5

In the High Barind Tract, in northwest

Bangladesh, a single crop of transplanted

rainfed rice (TPR), grown in the monsoon

aman season from June to October, provides a

major component of rural livelihoods.Aman

rice is vulnerable to late-season drought during

grain lling in October and in the rabi (winter)

season much of the land lies fallow. Cultivation

intensity in much of the Barind is considerably

less than in districts where irrigation allows

two or three rice crops to be grown each year.

Farmers lands are typically distributed over

a shallow sloping landscape or toposequence.

Two challenges of agricultural improvement

in such areas are to simultaneously improve

the reliability and yield of aman rice while

increasing total system productivity. Research

in recent years has demonstrated that these

objectives can be achieved through the

introduction of dry-seeded rice (DSR) and

the planting of short-duration rabi crops (e.g.,

mustard or chickpea) on residual moisture

immediately aer the rice harvest. Late onset

of the monsoon or low rainfall can delay rice

transplanting as a minimum of 600 mm of

cumulative rainfall is needed to complete land

preparation and transplanting. Dry seeding,

on the other hand, can be completed aer

land preparation by a power tiller aer much

less rainfall and the earlier planted DSR crop

matures 12 weeks before TPR, thus reducing

the risk of terminal drought, and allows earlier

planting of a following nonrice crop.

TPR requires less labor and dra power

for rice establishment than DSR, but the high

costs associated with weed control in DSR are a

major constraint to its adoption. Monitoring of

farmer-managed transplanted aman rice crops

in the Barind revealed that labor availability

constrains the timeliness of rst weeding for

Opportunities for direct seeding and improved weed controlin the Barind of Bangladesh

M.A. Mazid, C.R. Riches, A.M. Mortimer, and D.E. Johnson

many households and, with current practices,

34% of farmers lose more than 0.5 t ha1 of the

aainable yield because of weed competition

(Mazid et al 2001). Farmers in the Barind have

a strong preference for the late-maturing rice

cultivar Swarna. Use of this cultivar, however,

reduces the opportunity for establishing

chickpea or other rabi crops on residual

moisture, whereas growing earlier maturing

modern cultivars may contribute to an earlier

harvest. This report summarizes selected

ndings (aer Mazid et al 2003) from a long-

term eld experiment in the Barind designed to

explore the contribution of rice establishment

method, rice cultivar duration, and weed

control practices to aman rice performance and

the likely long-term impact on the composition

of the rice weed ora.

Methods

Rice establishment, nutrient management, and

weeding practices have been investigated on

farmland at Rajabari, Rajshahi, in northwest

Bangladesh from 2001 in an ongoing long-term

trial as described in greater detail in Mazid et

al (2001). The results for rice crops in 2000, 2001,

and 2002 are reported, comparing rice crop

establishment methods and weed management

practices. Treatments were (1) transplanted rice

(TPR)soil is puddled prior to transplanting;

the crop is hand-weeded twice at 30and 45days aer transplanting (DAT); (2) direct-seeded

rice (DSR)soil is plowed prior to seeding in

rows by hand, with hand weeding at 21, 33,

and 45 days aer sowing (DAS); (3) direct-seeded

rice with chemical weed control (DSRH)as for

DSR but with oxadiazon (375 g a.i. ha1) applied

24 days aer seeding,followed by one hand

weeding at 33 DAS. Plots of these treatments

were sown to the cultivarsSwarna (maturity


21/59

6

140145 days) and BRRI dhan39 (maturity

120125 days). Rice was harvested in 5-m2

plots. Biomass of individual weed species was

recorded in two unweeded quadrats per plot at

28 days DAS/DAT and total weed biomass at 45

DAS/DAT and again at harvest.

Results

Crop establishment method

With the exception of BRRI dhan39 in 2000,

yields from direct seeding of this and cv.

Swarna were as good as or beer than from

transplanting, the usual method of rice culture

in the district (Fig. 1). Early-season weed control

by preemergence application of herbicide

resulted in the highest yields, except for BRRI

dhan39 in 2000.

colona, Ecliptaprostrata, Eriocauloncinereum,

Fimbristylisdichotoma, Fimbristylismiliacea,

Hedyotiscorymbosa, Linderniaciliata, Ludwigia

sp.,Monochoriavaginalis, Paspalumdistichum,

and Sphaeranthusindicus. At harvest, there were

signicantly higher densities of weeds in DSR

(228 m2) than in TPR (75 m2; P 0.023). As

expected, however, at 45 DAS/DAT, the leastweed density and biomass were recorded in

DSRH. The range of responses by individual

weed species over three consecutive seasons

to crop establishment and weed management

practices is shown in Figure 2. An increase in

abundance (biomass at 28 DAS/DAT) of the

broadleaf speciesAlternantherasessilis, Eclipta

prostrata, Linderniaciliata, and Ludwigia sp. and

the sedges Cyperus diformis and Fimbristylis

miliacea was noticeable in DSR. Conversely, the

biomass ofMonochoriavaginalis was decreased

by direct seeding. The most noticeable increase

in abundance was seen in the perennial grass

Paspalumdistichum.

A long-term trial has demonstrated that,

although rice yield can be maintained with the

switch from transplanting to direct seeding,

farmers will face a greater weed problem

early in the crop season. Not only is there an

increased burden of weeds in direct-seeded

rice but the change in establishment practice

also leads to a shi in the relative abundance of

important species. Previous ndings indicate

that direct seeding was associated with higher

labor inputs for rst weeding than is the case

for transplanting. With the labor constraint, late

rst weeding, and a signicant yield gap due

to weeds on many farms in transplanted rice

with current weed control practices (Mazid et

al 2001), it is clear that the adoption of direct

seeding will need to be associated with the use

of chemical weed control. Studies suggest that

herbicides may nd a ready market in Rajshahi

District because (1) weeding is done almost

exclusively by hired labor and (2) the supply

of hired labor is local, with very lile weeding

done by seasonal migrant labor. Together, these

factors will create intense competition for labor,

especially on larger farms, which would be

intensied by the adoption of direct seeding.

Weed species shiftsThe weed ora of rainfed rice in the Barind

is diverse and exhibits high interseasonal

variability depending on water regimes at

rice establishment and soil moisture status of

toposequence position. Weed species present

in the experiment includedAlternanthera

sessilis,Ammaniabaccifera, Cyanotisaxillaris,

Cynodondactylon, Cyperusdiformis, Cyperusiria,

Cyperusrotundus, Cyperustenuispica, Echinochloa

5

4

3

5

4

3

DSR

DSRH

TPR

DSR

DSR

HTP

RDSR

DSR

HTP

R

000 00 00

B

A

Grain yield (t ha)

Fig. 1. Effect of establishment and weed control practices on the

yield (mean S.E.M) of rice cultivars BRRI dhan39 (A) and Swarna

(B). DSR = direct-seeded, hand-weeded; DSRH = direct-seeded +

herbicide; TPR = transplanted rice + hand-weeded.

Establishment method


22/59

7

Direct seeding advanced the rice harvest

by 7 to 10 days. Earlier harvest reduces the

problem of terminal drought in rice when

rains end abruptly in October and will ensure

that postrice crops are sown while seedbeds

are moist. Farmers with the least land under

cultivation (0.6 ha for the lowest quartile of

households) on average plant 43% to postrice

crops, oen on the least favorable land wheremoisture is limiting (Mazid et al 2003). Our

trials suggest that this group can maximize rice

yield and achieve timely planting of a high-

value chickpea crop by direct seeding rice. On

larger farms (> 2.5 ha for the upper quartile), a

lower proportion of land is planted aer rice,

using more favorable soils. Adoption of DSR by

this group could increase the area planted to

chickpea.

A single rice crop, combined with land

pressure and a high level of sharecropping

in the Barind Tract, leads farmers to place

a premium on optimizing rice yield and

household food security. This study

demonstrates that, although the widely grown

rice cultivar Swarna performs well under

direct seeding, the shorter duration BR39 is not

well adapted for this planting practice. Thisis because of the high levels of sterility in this

cultivar associated with owering during wet

periods. An earlier maturing variety with yields

to match those of Swarna could contribute

further to avoiding late-season drought during

grain lling and also allow earlier planting of

postrice crops. This challenge requires a broad-

based approach combining rice breeding with

agronomy and weed science.

Fig. 2. Effect of establishment and weed control practices on the biomass of nine rice weeds at 28 days after

planting in unweeded plots. See text for details.

9

DSR

DSRH

TPR

DSR

DSR

HTP

RDSR

DSR

HTP

R

7

5

3

7

5

3

5

3

9

5

3

0

5

0

5

50

30

0

8

6

4

5

0

5

00

50

00

50

Mean dry biomass g m- at 8 DAS/DAT

Alternanthera sessilis Cyperus difformis Cyperus iria

Eclipta prostrata Fimbristylis miliacea Ludwigia sp.

Lindernia ciliata Monochoria vaginalis Paspalum distichum

7

Establishment method


23/59

8

Information availability

With a shi to direct seeding, farmers will

be increasingly dependent on information

from outside sources. Indeed, successful

adoption and correct decision making

would be likely only if farmers had a greater

availability of current knowledge. Direct

seeding and associated weed managementcomprise not single recommendations but a

wide range of options that will be dependent

on toposequence, seasonal eects/rainfall

paern, the weeds present, and farmers

resources. Decision trees can provide farm-level

information in the form of structured questions

that enable answers in the form of options

to be chosen (Johnson and Mortimer 2004).

These trees specically focus on technical

issues related to the adoption of a particular

system and they may be useful tools to help

rene technology options for researchers and

extension sta. For a farmer in the favorable

DRUM-SEEDBROADCASTDRILL-SEEDBROADCAST

ARE SOIL CONDITIONS SUITABLE FOR

LINE SEEDING BY MACHINERY?

IS THERE A NEED FOR INTERROW CULTIVA-

TION OR SUBSTANTIAL HAND WEEDING?

ARE ANNUAL GRASSES ABSENT?

IS GOOD WATER MANAGEMENT POSSIBLE?

APPLY HERBICIDE

+ MANUAL WEEDING ORINTERROW CULTIVATION

APPLY HERBICIDE

+ LIMITED MANUAL WEEDING

DRY SEEDING

into a seedbed

WET SEEDING

sowing onto puddled saturated

soil

TRANSPLANTCAN FIELD BE DRY-CULTIVATED?

Is Cynodon dactylon or Cyperus rotundus ABSENT?

Yes No

Yes No

CAN FIELD BE DRAINED?

No YesNo Yes

Yes No

WEED MANAGEMENT

CROP ESTABLISHMENT

Fig. 3. Illustrative decision tree for the adoption of direct seeding with respect to favorable rainfed lowland rice.

rainfed environment, such as the Barind,

the question What are my options for rice

establishment? might initiate a tree involving

several steps and covering the range of direct-

seeding options (see Fig. 3 for an example). In

this example, the decision process recognizes

that a primary consideration will be the ability

of a farmer to drain his eld as only if this is

possible should direct seeding be recommended

because of the risk of early ooding. A second

decision level will involve the choice between

wet and dry cultivation and this will depend on

the ability to dry-cultivate and/or the presence

of perennial weeds. Further levels indicate

choices for row or broadcast seeding and

weed control options. Such a diagram allows

a structured approach to the range of options

available, and a more complex decision tree may

comprise weed control options for individual or

groups of weeds.


24/59

9

The transition to direct seeding and the

management of challenging weed problems

will require substantial information to enable

farmers to judge objectively what the best

technology options are. Gaining access to

such information may be a major obstacle

for potential adopters. The challenge for

researchers is to adequately address the

variability of the rice-farming systems for

which they are making recommendations

and to synthesize the results in ways that will

make the conclusions available to those who

will use them. Studies on direct seeding, weed

management, and decision tools are ongoing.

Acknowledgments

The research in Rajshahi was conducted by the

Bangladesh Rice Research Institute, NaturalResources Institute (UK), and University of

Liverpool in association with the Consortium

for Unfavorable Rice Environments (CURE),

and was partially funded under the Crop

Protection Programme by the Department for

International Development, UK.

References

Johnson DE, Mortimer AM. 2004. Issues for integrated weedmanagement and decision support in direct-seededrice. In: Rice is life: scientic perspectives for the 21stcentury. Proceedings of the World Rice ResearchConference held in Tokyo and Tsuukuba, Japan, 4-7November 2004. Los Baos (Philippines): InternationalRice Research Institute, and Tsukuba (Japan): Japan

International Research Center for Agricultural Science.CD. p 211-214.

Mazid MA, Jabber MA, Riches CR, Robinson EJZ, MortimerM, Wade LJ. 2001. Weed management implicationsof introducing dry-seeded rice in the Barind Tract ofBangladesh. Proceedings of the BCPC Conference Weeds 2001. 1:211-216.

Mazid M, Jabber MA, Mortimer M, Wade L, Riches CR,Orr AW. 2003. Improving rice-based cropping systemsin north-west Bangladesh: diversication and weedmanagement. Proceedings of the BCPC InternationalCongress on Crop Science and Technology 2003,SECC, Glasgow, UK. p 1029-1034.


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0

Purpose

This project aims to develop submergence-

tolerant cultivars that will improve the

livelihood and food security of farmers and

rice consumers in submergence-prone areas.

The major objective is to convert widely

grown varieties in rainfed lowland areas

into submergence-tolerant varieties through

incorporation of the Sub1 QTL on chromosome 9.

Background

Some 11 million hectares of shallow rainfed

lowland rice in South and Southeast Asia are

submergence-prone, and another 5 million

ha of medium-deep area experience stagnant

ooding of up to 50 cm. Submergence also

aects some of the areas classied as deepwater

(sustained water depths above 50 cm) on

around 4 million ha in Asia. Some estimates

indicate that submergence stress causes annuallosses of around US$1 billion in Asia (Dey and

Upadhyaya 1996, Herdt 1991). The normalized

yield loss (an index taking into account several

parameters) in submergence-prone areas

was estimated at about 80 kg ha1, causing a

production loss of about 3.2 million tons per

year, with a value of about $384 million. About

140 million people are at risk from ooding

damage in Bangladesh and ve states of

eastern India. With an average poverty ratio

of 45%, approximately 74 million poor peoplestand to benet signicantly from improved

submergence-tolerant rice cultivars.

Modern high-yielding rice cultivars are

seriously damaged if they are completely

submerged for a few days; however, a few

tolerant landraces were identied that can

withstand inundation for up to 2 weeks.

In these tolerant landraces, the Sub1 major

Breeding for submergence tolerance

D.J. Mackill, A.M. smail, S. Heuer, E. Septiningsih, A.M. Pamplona, R.M. Rodriguez,C.N. Neeraja, D. Sanchez, K. ftekhar, and G. Vergara

QTL accounts for most of the variation in

the trait. DNA markers linked to the Sub1

locus have facilitated its transfer into widely

grown cultivars that are locally adapted and

possess the quality aspects preferred by local

consumers. Those tolerant Sub1 cultivars have

considerably higher survival and yield under

submergence than susceptible cultivars.

Rice production can therefore be improvedand stabilized in submergence-prone areas

of South and Southeast Asia predominantly

inhabited by resource-poor farmers. The value

of submergence tolerance could be further

enhanced by combining it with tolerance for

medium-deep stagnant ooded conditions and

tolerance for submergence during germination.

Yield of these tolerant cultivars can be further

increased and stabilized through proper

management strategies.

Previous studies on submergence tolerance

The eects of ooding on rice as well as the

physiological bases of tolerance were recently

reviewed (Ram et al 2002, Jackson and Ram

2003). Plant survival in ooded areas depends

on various aspects of oodwater environments,

particularly the limitation of gas diusion,

irradiance level, and water temperature. Among

the important plant traits associated with

tolerance are high nonstructural carbohydrate

content before submergence, slowerunderwater shoot extension, optimum alcoholic

fermentation when O2 is low, an ecient

protective system upon air entry aer exposure

to low O2, and limited leaf chlorosis (Seer et

al 1997, Ram et al 2002, Jackson and Ram 2003,

Ella et al 2003). Carbohydrates remaining aer

submergence are necessary for recovery growth

and are correlated beer with survival than


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carbohydrate level before submergence (Das et

al 2005). A rapid regeneration growth following

submergence is essential under frequent or

prolonged ooding as this can ensure early

recovery and the production of sucient

biomass for high yield. Rice plants that exhibit

only limited elongation during submergence are

more tolerant of complete ooding and a strong

association between limited underwater shoot

growth and survival is commonly observed

(Jackson and Ram 2003, Das et al 2005).

Evidence is also accumulating for the role

of postsubmergence events in tolerance for

submergence. Tolerant cultivars acquired a

more ecient protective system to suppress

the level of active oxygen species and to lower

the extent of lipid peroxidation upon exposure

to air (Kawano et al 2002). Ethylene, a plant

hormone, accumulates in plant tissue during

submergence because of both enhanced

synthesis and entrapment, and this promotes

underwater leaf senescence. This eect is

suppressed in tolerant cultivar FR13A. Ella et al

(2003) found that blocking ethylene enhanced

chlorophyll retention and carbohydrate content

in the plant tissue and improved survival of

intolerant cultivar IR42.

Early ooding can cause poor crop

establishment in direct-seeded rice areas.Landraces tolerant of these conditions emerge

faster from the soil during ooding, produce

taller seedlings with more leaves, and aain

greater leaf area than intolerant lines. These

cultivars also maintain higher activity of

enzymes involved in the breakdown of starches,

for example, total amylases, under ooding and

this correlated positively with survival. Other

studies showed increased activity of some of the

enzymes involved in the fermentative pathways

during anaerobic conditions (Xie and Wu1989, Hossin et al 1996). Phosphofructokinase

(Fukao et al 2003) and pyruvate orthophosphate

dikinase (Huang et al 2005) were found to

be induced under low-oxygen stress, both of

which could enable substrate cycle operation of

adenosine tri-phosphates (ATPs) necessary for

energy production during germination.

Breeding improved submergence-tolerant

cultivars has been ongoing for more than

three decades (Mackill 1986, Mohanty and

Chaudhary 1986, Singh and Dwivedi 1996).

The initial work focused on transferring the

trait from traditional landraces into semidwarf

breeding lines. However, these lines were

low-yielding and had many undesirable

traits. Additional crosses resulted in the

development of tolerant breeding lines with

improved agronomic characteristics (Mackill

et al 1993, Mackill and Xu 1996, Mohanty et

al 2000). Although the lines with the highest

levels of tolerance had some yield penalty,

some breeding lines, such as IR49830-7, had

a yield equivalent to that of the irrigated

checks. These improved lines have been used

for further crosses. New breeding lines with

submergence tolerance were developed through

the Eastern Indian Rainfed Lowland Shule

Breeding Network (Singh et al 1998, Mallik et

al 2002). Some of these lines have been released

or recommended for release in India, such

as Kishori, Satyam, OR1234-12-1, CN 1035-61

(Bhudev), CRLC 899 (Varshadhan), TTB 238-3-

38-3 (Prafulla), NDR 8002, CR 2003-2, CR 2003-

3, CR 978-8-2, and IR54112-B2-1-6-2-2-2-CR2-1.

These lines together with Sub1 introgression

lines would serve as a basis for studies on crop

management and farmer participatory research.

Genetically, submergence tolerance is largelygoverned by a single major QTL, designated

Sub1, located on rice chromosome 9 (Xu and

Mackill 1996). Almost all strongly tolerant

cultivars possess the Sub1 QTL (Seer et al

1997); however, additional QTLs of smaller

eect appear to give increasing amounts of

tolerance (Nandi et al 1997). Through positional

cloning, a cluster of three putative ethylene

response factor (ERF) genes has been identied

in the Sub1 locus. ERFs are transcription factors

unique to plants, in which they constitute alarge multigene family related to Apetala2

(AP2) and dehydration-responsive element

binding (DREB) factors (McGrath et al 2005).

ERF genes are induced in response to several

biotic and abiotic stresses, and by ethylene and

other plant hormones, and might be involved

in cross-talk between the dierent pathways

(for review, see Guerson and Reuber 2004).

In addition, it was shown in rice and tomato


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that phosphorylation of the proteins enhances

DNA binding anity (Cheong et al 2003, Gu

et al 2000) and that ERFs act as transcriptional

activators and repressors, respectively (Fujimoto

et al 2000, Ohta et al 2001).

An allelic survey of the Sub1 ERF genes in

a range of tolerant and intolerant germplasm

revealed tolerant-specic alleles for Sub1A(Sub1A-1) and Sub1C (Sub1C-1). The tolerant

and intolerant alleles (Sub1A-2; Sub1C-2

to 8) show dierences in several putative

phosphorylation sites and are dierentially

expressed during submergence. Whereas

Sub1A-1 is highly expressed in tolerant lines

and expressed at a very low level in intolerant

accessions, Sub1C shows the opposite expression

paern. Overexpression of Sub1A-1 in an

intolerant variety conferred submergence

tolerance, suggesting that Sub1A-1 is the major

determinant of tolerance (Xu et al 2006).

The sequence information of these three

genes facilitated the development of ideal

markers suitable for backcrossing this locus

into widely grown varieties. The use of marker-

assisted backcrossing has been shown to be

eective in transferring Sub1 into a widely

grown Thai cultivar (Siangliw et al 2003).

In a previous project, the Sub1 genes were

introduced into widely grown varieties inthe rainfed areas of Asia. The sequences are

also being used to screen new landraces for

the presence of this gene. Preliminary data

indicate that some tolerant varieties such as

FARO 27 do not have the typical Sub1 gene

and might possess a dierent mechanism

of tolerance. Preliminary evaluation of Sub1

introgression lines showed that Sub1 can be

eective in conferring tolerance for 1014 days,

depending on oodwater conditions. However,

submergence sometimes occurs for a longerduration or more than once. Identication of

new sources of tolerance and genes additive to

Sub1 will therefore be highly desirable.

Recent studies at IRRI

The physiological basis of tolerance for ash

ooding is now reasonably well understood.

Two main factors were identied as being

important in contributing to injury when rice

is completely inundated: limited gas exchange

and reduced illumination. The consequences of

these are reduced underwater photosynthesis,

accelerated stem and leaf extension, and

enhanced chlorosis and leaf senescence,

resulting in a shortage in energy supply for

maintenance of metabolism. The mechanisms

by which tolerant cultivars depress the

damaging eects of submergence are becoming

more evident. Identication of the traits

associated with submergence tolerance will

help in designing ecient evaluation methods

to pyramid component traits and in gene

discovery. In addition, suitable management

strategies could be eciently designed and

tuned toward exploiting the potential of the

traits that are important for survival and

recovery.

Initial work at IRRI indicated that

submergence tolerance in the most tolerant

cultivars is mainly controlled by Sub1 and that

the most tolerant cultivars such as FR13A, Goda

Heenati, and Kurkaruppan all possess this

locus (Xu and Mackill 1996, Seer et al 1997, Xu

et al 2006). However, data suggest that other

genes are needed to gain higher submergence

tolerance. We have identied a tolerant-specic

allele of the Sub1C gene and have showndierential expression of this gene in tolerant

and intolerant accessions upon submergence.

It is currently unclear whether the relative

abundance of Sub1A and Sub1C gene products

is important for tolerance or whether Sub1

modulating factors are absent from intolerant

varieties. Such factors might be present in

additional minor QTLs controlling submergence

tolerance (Nandi et al 1997, Kamolsukyunyong

et al 2001) and could explain why some tolerant

breeding lines do not have as high a toleranceas FR13A.

Crosses were made with these lines for

further genetic and mechanistic analysis.

Breeders have been using the sources of

submergence tolerance such as FR13A and

Kurkaruppan to develop highly tolerant

cultivars with a high-yielding plant type.

The initial semidwarf breeding lines with

submergence tolerance similar to that of FR13A


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3

were developed in the late 1970s (described

in Mackill and Xu 1996). Breeding lines with

submergence tolerance from FR13A and high

yield potential were developed in the late 1980s

(Mackill et al 1993). More recently, breeding

lines developed from these sources have shown

promise in eastern India and a few of them

were released as new varieties.

Summary of recent achievements

Development of Swarna with submergencetolerance: A submergence-tolerant versionof Swarna was produced in 2 years by

marker-assisted backcrossing (MAB):

only the small fragment carrying the QTL

Sub1 was introduced. A large number

of BC1F1 seeds were produced. Based on

ve informative markers from the Sub1region, 54% of the plants were detected to

be Sub1+, out of which 3% (21 plants) had

desired recombination for the Sub1 region.

Background selection was performed

for the 12 chromosomes with 58 markers

(approx. 5 markers per chromosome). From

the progeny of 320 plants from the second

backcross, four plants were selected based

on foreground and background selection.

Out of 937 BC2F2 progeny from the selected

plants, one plant was selected for itsmaximum recipient genome and its target

locus.

Providing NARES partners withmultiplied seeds of Swarna-Sub1: Theseeds of Swarna-Sub1 have been multiplied

and used by CRRI, BRRI, and NDUAT for

phenotypic evaluation. In preliminary

yield trials at Rangpur, Bangladesh, and

Cuack, India, Swarna-Sub1 was evaluated

against other elite breeding lines andchecks. Swarna-Sub1 showed higher survival

(25% for Swarna vs. 100% for Swarna-

Sub1), a lower level of elongation under

submergence, and earlier maturity and it

did not lodge aer 10 days of submergence.

Data on yield and other aributes are being

analyzed. Swarna-Sub1 was evaluated for

adaptation to local conditions by growing it

in normal farmers elds at Cuack, Orrissa,

India, and Rangpur, Bangladesh. In India,

both Swarna and Swarna-Sub1 produced

similar yields in farmers elds (approx.

5.5 t ha1), whereas, at Rangpur, Swarna-

Sub1 slightlyoutyielded Swarna (3.9 t ha1)

and the local check Red Swarna (3.5 t ha1).

Swarna-Sub1 also had good plant height

and panicle number. Seeds of Swarna-Sub1

were multiplied at two sites in both India

and Bangladesh to produce sucient seeds

for large-scale testing in multiple farmers

elds in 2007 through participatory varietal

selection trials.

Identication of diagnostic markers for

precise MAB ofSub1: Previously, twocleaved amplied polymorphic markers

(CAPs) were designed targeting a silent

single nucleotide polymorphism (SNP)

located in the Sub1A gene and a unique

phosphorylation site in the Sub1C gene. To

more precisely measure the introgression

region of the Sub1 locus and to obtain

markers specic for functional genes

underlying the QTL, additional allele-

specic markers were developed. A second

SNP where the IR40931 allele causes an

amino acid change in the Sub1A protein(CCG in Teqing encoding for proline and

TCG in IR40931-33 encoding for serine)

was targeted for marker design. Since no

restriction enzyme sites were located at the

SNP locus that could be used to develop

a CAP marker, a dominant STS marker

was developed by designing a PCR primer

with the SNP at the 3 end. In addition,

several CAPs and indel (insertion/deletion)markers were also designed in the promoter

region of Sub1A and Sub1C. Several of thesegene-based markers have been used in our

MAB program, and have been found very

useful as alternative foreground markers.

The Sub1A marker has already been used

in CRRI, India, to conrm the presence of

the gene in some of their landraces and

improved varieties.


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4

Identication of additional microsatellite

markers for foreground/recombinantselection of the Sub1 locus: Additionalhighly polymorphic SSR primers from the

International Rice Genome Sequencing

Project (IRGSP) were identied in the region

of Sub1. Two of them were tightly linked

and located upstream of the Sub1 locus,

that is, RM23865 (6.2 Mb) and RM23869

(6.3 Mb). These two SSR markers have

been very useful in minimizing the size

of introgression of the Sub1 locus since

previously there were no tightly linked

polymorphic SSR markers located upstream

of the Sub1 locus.

Development of Samba Mahsuri and IR64with submergence tolerance: Previously,it was reported that Samba Mahsuri-Sub1

and IR64-Sub1 were produced within two

rounds of backcrossing and one generation

of self-pollination by MAB. Only the small

fragment around the tip of chromosome

9, where the QTL Sub1 is located, was

introduced into Samba Mahsuri, a popular

variety from India, and IR64. In addition,

another version of Samba Mahsuri-Sub1

with a smaller introgression of the Sub1

region was selected among 48 BC3F2progenies derived from a double-side

crossover type of BC3F1 plant. A similar

process was conducted for IR64. Seven

hundred BC3F2 plants derived from three

selected BC3F1 plants have been genotyped;

however, no double-side crossover has been

found. Nevertheless, several promising

recombinants having a smaller region of

Sub1 have been identied. We will genotype

the BC3F3 progenies of these recombinants to

identify the best plants having the smallestintrogression region of the Sub1 locus. This

plant can then be multiplied and used as an

alternative seed source of IR64-Sub1. Both

versions of Samba Mahsuri-Sub1 (BC2F2

and BC3F2) and IR64-Sub1 (BC2F2 and BC2F3)

have been used for seed multiplication.

A preliminary submergence eld test has

shown that Samba Mahsuri-Sub1 and IR64-

Sub1 were comparable with the tolerant check.

Development of TDK1 with submergencetolerance: Because of the small size ofthis population from the earlier backcross

and several markers that were biased

toward the tolerant parent, there were no

optimal plants to be selected out of the

BC2F2 population. However, several BC3F1

with very small Sub1 introgression regions

having no or one background introgression

have been identied. In addition, plenty of

BC3F2-derived BC3F1 seeds are available to

maximize the probability of nding the best

version of TDK1-Sub1. These BC3F2 plants

have been planted and will soon be ready

for genotyping.

Development of BR11 with submergencetolerance: A special case study was donefor BR11. Four selection strategies associated

with a marker-assisted backcross breeding

program were compared for validating

suitable selection strategies for an ecient

and eective MAB breeding program. The

four treatments related to the development

of BR11-Sub1 were as follows:

a. Foreground, recombinant, and

background selection

b. Foreground and phenotypic selection

c. Foreground, phenotypic, andbackground selection

d. Foreground, recombinant, and

phenotypic selection

As many as 1,430 BC1F1 plants were grown

from 44 F1 plants, and the best BC2F1 progenies

had been selected. Selection activities are

ongoing in the BC2F2 generation for treatment

b and in the BC3F1 generation for treatment

c. However, we could not advance the

selection processes a further generation fortreatment d because of the unavailability of

double-recombinant-type plants segregating

in the BC2F1 generation. Preliminary results

showed that phenotypic selection cannot be

used as an alternative for a marker-based

background in the MAB scheme.


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5

Development of CR1009 with submergencetolerance: DNA of CR1009 BC2F2 and BC3F1plants has been isolated and is ready for

genotyping.

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