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IV. Mechanisms of Controlled Pollination

IV. Mechanisms of Controlled Pollination

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43



(Freeman, 1970; Doggett, 1988). Flowering begins within 3 days of emergence of

the panicle from the boot. It starts at or near the apex and proceeds toward the

bottom of the panicle, being complete in 4 – 7 days. Schertz and Dalton (1980)

reported that stigmas are receptive up to 2 days before blooming and can remain

receptive for up to 16 days in the absence of pollination. Anthesis usually occurs

after sunrise but has been noted during the night hours, even as early as 10 p.m.

(Stephens and Quinby, 1934). Viable pollen is typically shed until about noon.

Based on these flowering habits, sorghum breeders have developed several

methods of pollination control. In fertile sorghum lines where self-pollinated seed

increases are desired, bagging the plant prior to flowering ensures self-pollination

and eliminates cross-pollination. To facilitate crossing, several methods of crosspollination have been developed, and each may be used to meet various

objectives within the breeding program. To create segregation for breeding and

selection, Schertz and Dalton (1980) suggested four methods for use in preparing the female flower for fertilization by the male parent: (1) hand

emasculation, (2) genetic male sterility, (3) hot-water emasculation, and (4) anther

dehiscence control by use of humidity. For commercial production of hybrid

cultivars, CMS is used. A brief description of each method and its basis for use

follows.



A. HAND EMASCULATION

Flowers are emasculated the day before anthesis. Such florets occur below

and within about 3 cm of opened florets in a sorghum panicle. All open

spikelets are removed with scissors. Panicles and equipment should be washed

to remove any pollen prior to emasculation, especially if the emasculation

occurs outdoors. There is less likely to be such pollen movement in greenhouses,

but such rinsing of panicles and equipment should be conducted to avoid

unwanted outcrossing.

All florets except those that are to be emasculated are removed, leaving only

the florets that are expected to open the next day. The three anthers are coaxed out

of the enclosing lemma and palea by inserting a sharpened pencil or similar

pointed instrument. Care must be taken not to break the anthers, and if the anther

is breached, that flower should be removed and instruments rinsed to avoid

contaminating the next floret. Every anther must be removed before the set of

florets is “completely emasculated.” The presence of one anther will cause

pollination of one or more ovaries prior to the transfer of pollen by the breeder.

After the florets are emasculated, a paper bag is placed over the emasculated

panicle until the florets are pollinated 1 – 2 days later. Field emasculation usually

is carried out during the afternoon in an attempt to avoid stray, viable pollen from

other plants.



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An experienced person can emasculate 10 –25 panicles per day (with each

panicle having 10 –20 emasculated florets). If more than one cross is desired with

the female parent, the panicle can be trimmed in such a manner that specific

sections of florets can be emasculated and pollinated 1– 2 days later. Regardless

of the number of emasculations per panicle, emasculated florets are pollinated by

collecting pollen from the male parent and then dusting it on the exposed stigmas

of the emasculated florets 1 –2 days after emasculation.



B. GENETIC MALE STERILITY

A series of nuclear recessive male sterility genes, designated as ms1 through

ms7, have been characterized in sorghum. These mutations, in the recessive

condition, result in a male-sterile plant that can be used for hybridization

(Rooney, 2001). Because these plants are completely male sterile, there is no need

to emasculate and larger numbers of seed can be made more easily. However, the

inability to produce true-breeding, uniform genetic steriles eliminates the use of

genetic male sterility for hybrid seed production. Consequently, genetic male

sterility has been used in sorghum-breeding programs to facilitate population

improvement programs in sorghum.

The use of genetic male sterility facilitates hybridization, but it also requires

close management of the population during anthesis. Once improvement is

completed, lines must be derived and the recessive ms alleles must be eliminated

or they will produce sterile progeny in the lines in future generations. Lines

segregating for genetic male sterility can be maintained by self-pollination of

random panicles or bulk pollination of sterile panicles with pollen from

heterozygous and male-fertile plants in the same row. To use this system, malesterile plants must be identified at tip flowering. Anthers in male-sterile plants are

smaller, thinner, and do not shed viable pollen. Upon identification, the tip of the

male-sterile panicle should be removed and the panicle bagged to avoid open

pollination. The panicle can then be pollinated 3 –5 days later with pollen

collected from the desired male parent. Hybrids from these crosses can be used

for population improvement or to begin another plant-breeding scheme, such as

pedigree selection for producing improved pure lines. Opportunities for the

utilization of genetic male sterility are well developed as breeders have

developed genetic male sterility stocks in many elite sorghum germplasms and

parental lines (Pedersen and Toy, 1997).



C. HOT- WATER EMASCULATION

Stephens and Quinby (1934) developed this method of emasculation to

produce a larger number of F1 seed prior to the availability of cytoplasmic male



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sterility. Open florets of the selected panicle are removed and the entire panicle is

enclosed in a waterproof sleeve of rubber or plastic tied securely around the

peduncle. The panicle is immersed in water heated to 42 –488C for 10 min. This

treatment kills the majority of the pollen grains but does not damage the ovary.

The head is allowed to dry and then covered with a paper bag. Pollen from the

selected male parent is dusted onto the sterilized panicle 3 –4 days after the hotwater treatment. Users of this approach should be aware that there will be a few

escapes (i.e., self-pollinations). This system is simple to use in the greenhouse,

but it is not commonly used today because other simpler methods such as

genetic male sterility and cytoplasmic male sterility have been identified

and developed.



D. CONTROL OF ANTHER DEHISCENCE CONTROL

Schertz and Clark (1967) developed a method to control anther dehiscence

using the humidity created from covering the panicle with a plastic bag prior to

flowering. This method, also known as plastic bag emasculation and/or poured

crossing, is commonly used to create segregating populations for breeding and

selection because it allows a breeder to make large numbers of crosses in a short

amount of time. Usually done in the field, plants selected for use as females in

poured crosses have flowered approximately 2.5 – 5 cm from the panicle apex.

The portions of the panicle that have flowered are removed (Fig. 1). The bottom

florets in the panicle are also removed, so that 3 –5 cm of the panicle remains.

This panicle is covered with a plastic bag and then covered with a pollinating

bag to shade the panicle and reduce the temperature under the plastic bag. The

bags remain on the plant for 2 – 3 days during which the panicle completes

anthesis. These bags create a highly humid atmosphere in which the moisture

content inhibits anther dehiscence. To complete pollination, pollen from the

male parent is collected, the plastic bag is removed, the head is “rapped or

jarred” to remove excess condensate and pendant anthers, and the pollination is

made immediately thereafter.

Because all of the anthers are not removed, a certain level of self-pollination

will occur in seed from a poured cross. In most cases, the proportion of progeny

that are F1 hybrids will vary based on the specific genotype used as a female

parent, the fecundity of the pollen parent, and the environmental conditions

during the process. To identify F1 hybrids, seed from the poured cross is planted

in a progeny row in the next generation. F1 hybrid plants must be identified by the

breeder on some specific phenotypic or genetic basis. This is typically

accomplished by using a simply inherited phenotypic trait or heterosis that

occurs between parents of disparate origin. This method should be avoided when

no markers are available and plants derived from selfing cannot be distinguished

from F1 plants.



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Figure 1 Photographic series which depicts the plastic bag emasculation process for making

breeding crosses in sorghum. (A) Panicle at mid-anthesis which is ideal for setting up the cross. (B) All

sections of the panicle which have already flowered are removed and any excess sections which will

not flower in the next 2 days are removed from the lower part of the panicle. (C) The cut panicle is

bagged with a plastic bag, and covered with a paper bag. (D) Two to three days later, pollen from the

male parent is collected, the paper bag is removed and the female panicle is rapped to remove excess

anthers. The plastic bag is removed and the panicle is pollinated immediately. (E) Seed from the cross

is grown the following generation and in this row hybrids are easily detected by heterosis and seed

color (F1s are red while female parent is white).



E. CYTOPLASMIC – GENETIC MALE STERILITY

Unlike the methods previously described, the CMS system is not used for

population development, but CMS is the mechanism that makes the production of

the hybrid sorghum seed economically feasible. The CMS system relies on a set

of male sterility-inducing cytoplasms that are complemented by alleles at genetic



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Table I

Summary of the Different Cytoplasmic–Genetic Male Sterility Systems Identified

in Sorghum bicolor L. Moench

Cytoplasm

Group

A1



A2



A20

A3

A4



Source



Restorer source



Genetics of restoration



Simple, dominant,

Numerous,

including Tx430,

sporophytic

Tx7078

IS12662C Numerous,

Simple, dominant,

including IS2801C,

sporophytic

Tx430

IS3063C, IS2801C

Simple, dominant,

IS1056C

sporophytic

IS1112C, IS1112C

Simple, two genes,

IS12565C

gametophytic

IS7920C IS2801C

Unknown



Milo



Reference

Worstell et al. (1984)

and Klein et al. (2001b)

Worstell et al. (1984)



Worstell et al. (1984)

Worstell et al. (1984)

and Tang and Pring (2003)

Worstell et al. (1984)



loci in the nuclear genome that either restore fertility or maintain sterility. There

are many different CMS systems documented in sorghum, each caused by a

different mutation in the cytoplasm and each is complemented by different

nuclear restoration loci (Table I). For most CMS systems the interaction of

cytoplasmic and nuclear genes defines whether all specific lines are fertile or

sterile. In the CMS system, lines that have [A] cytoplasm must have a dominant

allele present in the nuclear genome to restore male fertility (Table II). If the line

lacks the dominant allele for fertility restoration, the plant will be male sterile.

The genetic factors for each CMS system are inherited independently of each

other. It is possible for a single line to restore more than one CMS system. The

frequency of lines capable of restoring fertility varies among with specific race

and method of restoration. The most commonly used CMS system is the A1

system. This was the original CMS system identified and characterized by

Table II

Genotypes and Corresponding Phenotypes for A-, B-, and R-Lines in the A1

Cytoplasmic–Genetic Male Sterility System in Sorghum

Line



Cytoplasma



Genotypeb



Phenotype



A-line

B-line

R-line

Hybrid



[A]

[N]

[A] or [N]

[A]



rf rf

rf rf

RF RF

RF rf



Male sterile

Male fertile

Male fertile

Male fertile



a

b



Cytoplasm types: [A], sterility-inducing cytoplasm type; [N], normal cytoplasm.

Genotype: RF, dominant allele for fertility restoration; rf, recessive allele for fertility restoration.



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Stephens and Holland (1954) and further characterized by Maunder and Pickett

(1959). The vast majority of commercial hybrid seed production uses A1

cytoplasm. Of the remaining systems, only a limited amount of hybrid seed has

been produced using the A2 system.

Hybrid seed production requires maintenance of the A-, B-, and R-lines

(Fig. 2). Seed of a male-sterile A-line is increased by pollination using the

complementary B-line. The sole purpose of the B-line (also known as a

maintainer) is to perpetuate or maintain the A-line. The A-line and B-line are



Figure 2 Schematic of the sorghum hybrid seed production process utilizing cytoplasmic–

genetic male sterility. The A-line parent is increased utilizing pollen from the B-line. The F1 hybrid is

produced by pollinating the A-line with an R-line pollinator. That seed is sold to producers for grain

production. Both the B-line and R-line are maintained through self-pollination. The inset picture is of a

hybrid seed production field in which the red parent is the female and the white parent is the pollinator.



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isocytoplasmic, meaning that these two lines are genetically identical except that

the A-line has a sterility-inducing cytoplasm while the B-line has normal fertile

cytoplasm. Thus, A-line plants that are male sterile can be pollinated with pollen

from B-line plants to regenerate seed of the A-line which is 100% male sterile.

To produce hybrid seed, the male-sterile A-line is pollinated with pollen from the

male-fertile R-line plants. The R-line (also known as a restorer line) is genetically

very different than the A-line and it carries the dominant fertility restoration

alleles needed to restore fertility in the progeny of the A-line. The seed that is

produced on the A-line from this pollination is the seed that is planted by the

producer for commercial grain production.

In the early stages of hybrid development, these crosses usually are made

using pollination bags and hand transfer of pollen, and fertile lines are maintained

by bagging to ensure self-pollination (Fig. 3). Upon the commercialization of a

hybrid, seed increases are made in environments that are conducive to seed

production and quality. Typically, 12– 18 rows of the male-sterile A-line are

planted with 2 –6 rows of the pollinator, R-line, interspersed between sets of

A-line rows (Fig. 2). The exact ratios of female to male rows vary depending on

the company and producer preferences. The male rows may be harvested early or

they may be cut down to eliminate contamination prior to harvest of the crop. In

addition, the rows are typically rouged several times to eliminate off-type plants

in the seed increase. At maturity, the seed on the A-line is harvested, cleaned,

treated, and bagged for commercial sale to producers.



V. IMPROVEMENT METHODOLOGY

As mentioned previously, efforts to improve sorghum have led to significant

changes in the types of sorghum that are currently grown. In the early 20th

century, sorghum improvement switched from farmer selection to trained plant

breeders. Until 1956, sorghum breeders selected and developed pure-line

cultivars that were grown by producers. Therefore, sorghum breeders followed

the breeding procedures developed for self-pollinated crops. After 20 years of

development, hybrid sorghums were first marketed in 1956 and adoption of

hybrids in the US was nearly 100% only 5 years later. In most of the developed

world, hybrid sorghums comprise the vast majority of production and in these

regions breeders switched their emphasis to hybrid development. In this situation,

many of the techniques developed for corn breeding were now applicable to

sorghum hybrid-breeding programs. Alternatively, in many of the less developed

regions of the world, sorghum producers still rely on pure-line cultivars.

Therefore, sorghum breeders must use methodology appropriate for the

development of either pure lines or hybrids.



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Figure 3 Self-pollinated (A) and experimental testcross hybrid (B) increases of sorghum in a

breeding program. Self-pollination ensures the purity of the parental lines as some level of outcrossing

occurs naturally. In the development of experimental testcross hybrids, pollen is moved from the

R-line row directly onto the A-line row to the right. Testcrosses are identified by the striped bags.



Because of the variation in types of sorghums grown throughout the world,

numerous breeding procedures have been developed and adopted by sorghum

breeders. Since sorghum is a self-pollinated species, most of the breeding

methodologies (both cultivar and hybrid) are based on the production of

segregating populations which is followed by selection in segregating

populations. The selections are usually allowed to self-pollinate during selection

to produce homozygous uniform lines. Where pure-line cultivars are grown, the



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potential of these new lines is evaluated, while in hybrid-breeding programs these

lines will be testcrossed to measure their value as a parental line. Population

improvement program efforts are also being undertaken in a few sorghumbreeding programs. This type of approach is facilitated by access to usable

sources of genetic male sterility. The commonly employed methodologies for

sorghum improvement are described in more detail.



A. POPULATION IMPROVEMENT

The goal of most population improvement programs is to accumulate

favorable alleles for the traits of interest while maintaining as much genetic

diversity as possible. These recurrent selection methods usually require

significant amounts of hybridization. In the past, there was relatively little

effort in population improvement in sorghum due to the fact that sorghum is a

self-pollinated species. The integration of genetic male sterility into adapted

germplasm has facilitated their adoption into some sorghum-breeding programs

and for specific applications. While population improvement programs are not

the dominant method of sorghum breeding, they are useful as a source of

genetic variation and improved traits.

In a population improvement program, genetic male sterility eliminates the

need for emasculation. The most commonly used genetic male sterile is ms3

(Rattunde et al., 1997). The method of selection in population improvement

programs in sorghum ranges from mass selection to family-based selection. In

mass selection, breeders select individual plants expressing the trait of interest and

then all the seed from the selected plants are bulked. To maintain segregation and

recombination in the next cycle, sorghum breeders must hybridize selections or

select from male-sterile plants. In family-based population improvement

programs, families are created and then these families are evaluated in replicated

testing to identify the most suitable parental genotype. The breeder uses this

information to select the parental lines used to create that family. Like mass

selection, the breeder must intermate the selections using controlled pollination or

genetic male sterility to produce the populations for the next cycle. Various family

types are evaluated ranging from half-sib, full-sib or S1 families (Hallauer and

Miranda, 1981).

Sorghum-breeding programs have used population improvement for a wide

variety of traits including drought tolerance, increased yield, wide adaptation,

improved quality, and pest resistance. Significant improvements in yield have

been reported using this methodology, but the transfer of the gains made in

population improvement programs to hybrids has been difficult (Rattunde et al.,

1997). Typically, germplasm from population improvement programs must be

self-pollinated to produce inbred and uniform lines that are acceptable for hybrid

production. If genetic male sterility was used in the population improvement



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program, then the breeder must take care to ensure that the recessive alleles that

cause male sterility are eliminated from the derived lines. Once uniform, all

materials from a population improvement program must pass through an inbred

and pure-line development program to be used in commercial production.



B. CULTIVAR AND INBRED LINE DEVELOPMENT

Regardless of the breeding goals, sorghum-breeding programs, must at some

point, produce inbred or pure lines. The specific approaches vary and may include

breeding methods such as pedigree, bulk, and single-seed descent, but they all

allow for self-pollination. In general, specific crosses are made using the

methodology described previously between lines for the specific improvement of

certain traits. F1 progeny are self-pollinated to produce an F2 population. From

the F2 generation until uniform lines (usually between the F4 and F6 generation)

are produced, sorghum breeders use various methods of selection to improve the

agronomic, disease, and stress characteristics of these lines (Fig. 4). Each

program has standards for evaluating these lines for specific characteristics at

defined generations. The appropriate generation for the selection of specific traits

is dependent on the heritability of the trait and the environments in which the

sorghum breeder is selecting. In general, traits with higher heritability (maturity,

height, grain color, etc.) are selected in the early generations while traits with

lower heritability are selected in more advanced generation (yield, drought

tolerance, disease, and insect resistance). These more complexly inherited traits

must also be screened in specific environments, where they may or may not be

expressed in any given year. As the lines approach phenotypic uniformity, pureline breeding programs begin replicated evaluation and agronomic testing. In

hybrid programs, the new lines are testcrossed to confirm whether the line

restores or maintains fertility to measure their general combining ability and

suitability as a parent in hybrids.



C. HYBRID DEVELOPMENT

Long before sorghum hybrids were a reality, sorghum breeders were aware of

the potential yield increases offered in hybrids. Stephens and Quinby (1952)

documented the yield advantages of hybrid sorghums using hybrids produced

by hot-water emasculation techniques. The limiting factor to hybrid production

was the lack of an economically feasible method of producing hybrid seed.

The development of the CMS system eliminated this problem and sorghum

hybrids were adopted immediately upon their commercial release in the late

1950s. Quinby et al. (1958) reported yield increases of 58 and 22% over the best

parent under dryland and irrigated conditions, respectively. In a study of 391

locations in four countries, Doggett (1969) found that hybrid yields were



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Figure 4 Pedigree breeding scheme used by the Texas Agricultural Experiment Station sorghumbreeding program at College Station, Texas. This scheme is used for the development of new B- and

R-line germplasm. Initial crosses are made using either plastic bag crosses or hand emasculations.

Open-pollinated selections are made in each generation until the F5 where the plot is self-pollinated

and used to make testcross hybrids. At the F5 generation, new B-lines enter sterilization and

testcrossing while new R-lines are evaluated in testcrosses.



consistently higher than that of the best parent and the advantages of hybrids were

accentuated in dryland environments. Sorghum breeders have been able to

improve the productivity of hybrids as well. Miller and Kebede (1984) reported a

40% yield increase in new hybrids over the original sorghum hybrids of the late

1950s. While hybrid productivity was increasing, it is critical to note that

the productivity of the inbred parents increased as well (Miller and Kebede,

1984). These trends clearly indicate the importance of both additive and

dominant gene action in sorghum.



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