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Hybrid seed production requires a male sterile parent. Why?

Hybrid seed production requires a male sterile parent. Why?


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Hybridization is the process of crossing two plant species or varieties. In this case, two varieties of plants. In plant breeding, crossing is a very useful technique to improve the features of the offspring. For example, if a maize corn variety has lost yield over the years, it can be crossed to a variety with a higher yield and the offspring will also have a higher yield.

In order to cross these two varieties, the male needs to be sterile. Why is that? I don't understand how the process works and I cannot find the reason anywhere. The answer should be pretty basic, and I think I am missing something that everybody assumes I should know.


male-sterility is not required for breeding.

Male-sterility means that the female acting plant (the plant that will bear the fruit) has sterile male organs (either dysfunctional anthers or sterile pollen). and won't self pollinate. The male-acting plant is fertile.

In monoecious plants this is helpful for breeding because we can't easily provide a controlled pollinating environment by separating the female and male organs

Tomatoes are monoecious (hermaphrodites), self-pollinated. The pollination occurs (usually) before the flower opens. Male-sterility prevents pollination and allows the breeder to pollinate.

Corn is a cross-pollinated plant with some capacity for self-pollination, male-sterility in corn helps ensure that the corn plant is not pollinated by itself.

Male-sterility gives peace of mind and easier breeding techniques but is not a requirement.

You can expand your knowledge using Principles of Plant Genetics and Breeding, Second Edition by George Acquaah. Check chapters 5: Introduction to Reproduction and Autogamy. Searching "male sterility in plants explained" in google yielded many relevant pdfs.


To get a hybrid you need pollen from a different plant. Corn is likely to self-pollinate if some action is not taken to prevent it. I don't know of sterile males plants - in the US corn belt the self-pollination is prevented by detasseling. Many young people get summer jobs detasseling (cutting off the tassels) for seed corn.


Joseph Gottlieb Kölreuter was the first to document male sterility in plants. In the 18th century, he reported on anther abortion within species and specific hybrids.

Cytoplasmic male sterility (CMS) has now been identified in over 150 plant species. [2] Male sterility is more prevalent than female sterility. This could be because the male sporophyte and gametophyte are less protected from the environment than the ovule and embryo sac. Male-sterile plants can set seed and propagate. Female-sterile plants cannot develop seeds and will not propagate.

Manifestation of male sterility in CMS may be controlled either entirely by cytoplasmic factors or by interactions between cytoplasmic factors and nuclear factors. Male sterility can arise spontaneously via mutations in nuclear genes and/or cytoplasmic or cytoplasmic–genetic. In this case, the trigger for CMS is in the extranuclear genome - (mitochondria or chloroplast). The extranuclear genome is only maternally inherited. Natural selection on cytoplasmic genes could also lead to low pollen production or male sterility.

Male sterility is easy to detect because a large number of pollen grains are produced in male fertile plants. Pollen grains can be assayed through staining techniques (carmine, lactophenol or iodine).

Cytoplasmic male sterility, as the name indicates, is under extranuclear genetic control (under control of the mitochondrial or plastid genomes). It shows non-Mendelian inheritance , with male sterility inherited maternally. In general, there are two types of cytoplasm: N (normal) and aberrant S (sterile) cytoplasms. These types exhibit reciprocal differences.

While CMS is controlled by an extranuclear genome, nuclear genes may have the capability to restore fertility. When nuclear restoration of fertility genes is available for a CMS system in any crop, it is cytoplasmic–genetic male sterility the sterility is manifested by the influence of both nuclear (with Mendelian inheritance) and cytoplasmic (maternally inherited) genes. There are also restorers of fertility (Rf) genes that are distinct from genetic male sterility genes. The Rf genes have no expression of their own unless the sterile cytoplasm is present. Rf genes are required to restore fertility in S cytoplasm that causes sterility. Thus plants with N cytoplasm are fertile and S cytoplasm with genotype Rf- leads to fertiles while S cytoplasm with rfrf produces only male steriles. Another feature of these systems is that Rf mutations (i.e., mutations to rf or no fertility restoration) are frequent, so that N cytoplasm with Rfrf is best for stable fertility.

Cytoplasmic–genetic male sterility systems are widely exploited in crop plants for hybrid breeding due to the convenience of controlling sterility expression by manipulating the gene–cytoplasm combinations in any selected genotype. Incorporation of these systems for male sterility evades the need for emasculation in cross-pollinated species, thus encouraging cross breeding producing only hybrid seeds under natural conditions.

Hybrid production requires a plant from which no viable male gametes are introduced. This selective exclusion of viable male gametes can be accomplished via different paths. One path, emasculation is done to prevent a plant from producing pollen so that it can serve only as a female parent. Another simple way to establish a female line for hybrid seed production is to identify or create a line that is unable to produce viable pollen. Since a male-sterile line cannot self-pollinate, seed formation is dependent upon pollen from another male line. Cytoplasmic male sterility is also used in hybrid seed production. In this case, male sterility is maternally transmitted and all progeny will be male sterile. These CMS lines must be maintained by repeated crossing to a sister line (known as the maintainer line) that is genetically identical except that it possesses normal cytoplasm and is therefore male-fertile. In cytoplasmic–genetic male sterility restoration of fertility is done using restorer lines carrying nuclear genes. The male-sterile line is maintained by crossing with a maintainer line carrying the same nuclear genome but with normal fertile cytoplasm.

For crops such as onions or carrots where the commodity harvested from the F1 generation is vegetative growth, male sterility is not a problem.

In hybrid maize breeding Edit

Cytoplasmic male sterility is an important part of hybrid maize production. The first commercial cytoplasmic male sterile, discovered in Texas, is known as CMS-T. The use of CMS-T, starting in the 1950s, eliminated the need for detasseling. In the early 1970s, plants containing CMS-T genetics were susceptible to southern corn leaf blight and suffered from widespread loss of yield. Since then, CMS types C and S were used instead. [3] Unfortunately, these lines are prone to environmentally induced fertility restoration and must be carefully monitored in the field. Environmentally induced, in contrast to genetic, restoration occurs when certain environmental stimuli signal the plant to bypass sterility restrictions and produce pollen anyway.

Genome sequencing of mitochondrial genomes of crop plants has facilitated the identification of promising candidates for CMS-related mitochondrial rearrangements. [4] The systematic sequencing of new plant species in recent years has also uncovered the existence of several novel nuclear restoration of fertility (RF) genes and their encoded proteins. A unified nomenclature for the RF defines protein families across all plant species and facilitates comparative functional genomics. This nomenclature accommodates functional RF genes and pseudogenes, and offers the flexibility needed to incorporate additional RFs as they become available in future. [5]


Single-cross Hybrids

A hybrid plant results from a cross of two genetically different plants. The two parents of a single-cross hybrid, which is also known as a F1 hybrid, are inbreds. Each seed produced from crossing two inbreds has an array (collection) of alleles from each parent. Those two arrays will be different if the inbreds are genetically different, but each seed contains the same female array and the same male array. Thus, all plants of the same single-cross hybrid are genetically identical. At every locus where the two inbred parents possess different alleles, the single-cross hybrid is heterozygous.

Plants of a single-cross hybrid are more vigorous than the parental inbred plants. In Figures 2a and 2b, the single-cross hybrid plant and ear are shown with the plants and ears of the parental inbreds. Clearly, the hybrid plant is taller and the hybrid ear is larger. The increase in vigor of a hybrid over its two parents is known as hybrid vigor.

Figure. 2a: Corn Plants: Inbred B73 (left), Inbred Mo17(middle), Single cross B73 x Mo17 (right) (UNL, 2004)

Figure. 2b: Corn Ears: Inbred B73 (left), Inbred Mo17(right), Single cross B73 x Mo17 (middle) (UNL, 2004)

Breeders often measure the degree of hybrid vigor of a trait with the following formula:

where Hyb = the value of the trait in the hybrid and

MP = the average (mid-parent) value of the trait in the two parents. For For example, in Figure 2a the height of the single-cross hybrid is 3.0 m (this equals Hyb), the average height of the inbreds is 2.0 m (this equals MP), and the value of hybrid vigor is 50%. Hybrid vigor calculated in this way is called mid-parent hybrid vigor. Another type is high-parent hybrid vigor. This is the superiority, expressed as a percentage, of the hybrid over the parent with the better or higher value of the trait. Corn breeders will be successful in increasing hybrid performance if the hybrid vigor of a new hybrid compared to an older hybrid is increased and the two sets of parents have equal performance and/or if hybrid vigor is unchanged but the mid-parent value of the parents of the newer hybrid is superior to that of the parents of the older hybrid.

The genetic basis of hybrid vigor is not completely understood. However, experience has shown that a hybrid produced by crossing two inbreds that are closely related usually will exhibit less hybrid vigor than a hybrid produced by crossing inbreds that are more distantly related.

If a single-cross hybrid is allowed to open-pollinate (pollen is dispersed freely), each of the plants grown from the resulting seed will be genetically unique. To understand why this is so, first consider a single locus. All plants of a single-cross hybrid are genetically identical, so at a single heterozygous locus any cross- or self-pollination occurring with open-pollination can be represented as:

One-half of the egg cells produced by each plant carries the A1 allele and one-half carry the A2 allele. The same is true of the pollen cells. The egg and pollen cells then combine at random during pollination.

The A1A2 and A2A1 genotypes are functionally identical, so three unique genotypes can be produced at a single heterozygous locus when open-pollination occurs.

With two heterozygous loci, A and B, the situation can be illustrated as follows:

In the two-locus case, nine unique genotypes are produced. This occurrence of multiple genotypes among progeny arising from the self- or cross-pollination of parents that all have the same heterozygous genotype at one or more loci is known as genetic segregation.

This segregation occurs at hundreds or even thousands of gene loci. The number of unique genotypes resulting from genetic segregation at n loci is given by 3 n . Thus, if n=1 (i.e., one locus), then the number of unique genotypes is three, and if n=2 the number of unique genotypes is nine. But, if n=20, the number of unique genotypes balloons to 3,486,784,401 (=3 20 ) Any commercial single-cross hybrid of corn is likely heterozygous at many more than 20 loci. That is why open-pollination of such a single cross results in progeny that are all genetically unique.

The progeny produced from self-pollination of a F1 single-cross hybrid are known as F2 plants. On average, F2 plants will have vigor that is approximately half-way between the single-cross parental plants and the average of the two inbred grandparents that is, half of the hybrid vigor is lost. This is illustrated in Figure 3. The F2 ears on the bottom row vary in size, but on average are larger than the ears from their inbred grandparents and smaller than the ear from their single-cross parent. That is why farmers have an incentive to purchase new single-cross hybrid seed each year.

Figure. 3: Top row of corn ears: Inbred B73 (left), Single cross B73 x Mo17 (middle), Inbred Mol7 (right) Bottom row of corn ears: From F2 plants derived from B73 x Mo17 (UNL, 2004)

When single-cross hybrid seed is commercially produced, one inbred is the male parent and the other the female parent. Either the female parent must be male-sterile (pollen is not produced or is not functional) or the tassel on each female plant must be removed (this is called detasseling) prior to any pollen production (Figure 4). In either case, all the seed produced on the female parent will be single-cross hybrid seed.

Figure. 4: A single-cross hybrid production field with female inbred parent detasseled) and male inbred parent (not detasseled) (UNL, 2004)

Developing an inbred from a single-cross hybrid requires approximately seven generations of repeated self-pollinations (Figure 1). Each year in the United States, commercial seed companies produce hundreds of new inbreds and test in field trials many thousands of new single-cross hybrids obtained by crossing these inbreds. Compared to existing commercial hybrids, the vast majority of these new hybrids will be poorer or no better in performance. Only the hybrids that have superior performance in these trials are produced in mass quantities and sold as commercial hybrids to farmers.

Considerable time and inputs are required to develop, select, and produce single-cross hybrids. Achieving a high level of cost efficiency of these processes typically requires large-scale operations.


A new cytoplasmic male sterility system for hybrid seed production in Indian oilseed mustard Brassica juncea

We report a novel cytoplasmic male sterility (CMS) system in Brassica juncea (oilseed mustard) which could be used for production of hybrid seed in the crop. A male sterile plant identified in a microspore derived doubled haploid population of re-synthesized B. napus line ISN 706 was found to be a CMS as the trait was inherited from the female parent. This CMS, designated ‘126-1’, was subsequently transferred to ten different B. juncea varieties and lines through inter-specific crosses followed by recurrent backcrossing. The F1s of inter-specific crosses were invariably partially fertile, but irrespective of the variety/line used, the recipient lines became progressively male sterile over five to seven generations and could be maintained by crossing the male sterile lines with their normal counterparts. The male sterile lines were found to be stable for the trait under both long and short day conditions. CMS lines when crossed with lines other than the respective maintainer line were restored for fertility, implying that any variety could act as a restorer for ‘126-1’ cytoplasm in B. juncea. These unique features in maintenance and restoration of CMS lines coupled with near normal floral morphology of the CMS lines have allowed the use of ‘126-1’ cytoplasm for hybrid seed production. The uniqueness of ‘126-1’ has been further established by Southern hybridization with mitochondrial DNA probes and by a histological study of the development of male sterile anthers.

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Discussion

In this study, we have developed a male sterility–fertility restoration system for heterosis breeding in plants. In the female parent, tapetum-specific, high-level and postmeiotic expression of the Arabidopsis BECLIN1/ATG6 gene led to complete male sterility. The tapetum–specific expression was completely abolished and male fertility was restored in the F1 hybrid.

The male sterility system (construct 1371 Supplementary Fig. S1a-I) is the modification of our previously reported two component system 15 based on the principle of expression restoration of a TGTA-mutated promoter by providing a complementing TBPm3, which binds specifically to the TGTA 36 . The system gave several-fold enhancement of expression over the native tapetum-specific promoter (Fig. 1a), in agreement with our previous report 15 . In this study, we included the light-regulated transcription factor HFR1 that exhibits its COP1-mediated degradation 37,49,50 to limit TBPm3 and abolish expression of the desired gene. HFR1 is a transcription factor (bHLH) consisting of 292 amino acids, of which the N-terminus 131 amino acid interacts with COP1 and the C-terminus 161 amino acid has a functional role to bind DNA and promote photomorphogenesis 39,49 . We fused the HFR1 NT131 fragment to the N-terminus of TBPm3, thus making the fusion protein HFR1 NT131 -TBPm3. This fusion did not affect the functionality of TBPm3, resulting in a high-level expression of the desired gene (gusA or BECLIN1) (Fig. 1a). The transgenic plants expressing gusA were normal in growth and development and the strength and stringency of the expression system were not compromised (Fig. 2b,c–f and Supplementary Fig. S1-b and c). To ensure the transcriptional abolition of tapetum-specific expression in F1 progeny, it was crossed with the male parent expressing COP1 under the regulation of the Arabidopsis P A9 promoter 47 (Fig. 1b). The expression of COP1 ensures its availability to bind with HFR1 NT131 and to degrade it in the F1 tapetal cell. The degradation of TBPm3 was also achieved, as it was conjugated HFR1 NT131 -TBPm3 (Fig. 1c). However, COP1 facilitates only partial abolition of the TGTA-TBPm3 complementation system in F1 (Fig. 2b,c–f). The COP1 protein is known to shuttle between the cytoplasm and nucleus 39 and stoichiometrically insufficient nuclear concentration of COP1 might be a reason for partial expression reversion. Therefore, we made use of an alternative COP1-mutant (COP1 L105A ) 38 . The mutation retained dimerization and functional activity of the COP1 but increased its nuclear abundance. The expression of the reporter gene (gusA) was completely abolished in F’1 progeny when COP1 L105A lines were used as a male parent, instead of native COP1 (Fig. 2b,c–f). Thus, a tapetum-specific reversible expression system was established.

The male sterility and fertility restoration system developed by us are generic in nature and hence can be used with other reported genes for male sterility. Thus, the female expression cassette (1371) can be used to generate the male-sterile parent by expressing any known gene reported for the male sterility such as BARNASE 18 , BAX 28 and so on. The major advantage with our system that there is no specific requirement of the fertility restoration gene for example BARSTAR in BARNASE 18,19 . The male expression cassette (Construct 1373) offers restoration through abolishing transcription of the male sterility gene. We used the Arabidopsis BECLIN1/ATG6 15 gene to raise complete male sterile transgenic plants. Genetic engineering of male-sterility and the fertility-restoration system have emerged as tangible options for hybrid seed production. Several restoration systems have been reported to redeem male fertility by inactivating the male sterility protein 18,19 , degrading transcripts of the male sterility gene 14,17,51 and site-specific recombination in the male sterility gene 8,10 . However, efficient restoration of fertility has been discussed as one of the limiting factors in some of the systems 14,21,51 . The present system offers a system equipped with complete male sterility and fertility restoration in F1-progeny (Figs 4,1a–c proposed model) with a novel approach.

The restoration of fertility of F1 hybrid is prerequisite, especially when the economic product is seed. The barnase/barstar system 18,19 was deployed for the commercial hybrid production but the identification of efficient restorer (barstar) line was proven to be difficult in Brassica juncea one in 54 cross-recombination between barnase (male sterile) × barstar (restorer) was adequately restored male fertility in the F1 hybrid 21,23 . Tapetum-specific promoter TA29 driven barstar (restorer) restore 65.6% male fertility (in terms of pollen viability), it was further improved to 78–90% when P A9 and chimeric system were used to express barstar (restorer) 9,21 . Barnase weakly expressed in vegetative tissue resulted yield penalty in the plants 23 . The other barnase based systems the Cre/loxp-mediated site-specific recombination system 10 , two-component system 22 and split-gene system 4,11 claimed 100% restoration of F1 fertility, however, use of toxic gene of trans-origin limited the acceptability due to biosefty concern in some countries. Pathogenesis-related (PR) β-1,3-glucanase gene based male sterility was only partially restored by pA9-driven sense and antisense PR glucanase fragments 51 . The temperature-sensitive DIPTHERIA TOXIN-A (DTA ts ) confer conditional-male-sterility (18 ° C male sterility, 26 ° C restored fertility) 32 and reversible male sterility in egg plant 16 claimed complete restoration but works on ethanol inducible method which limit its practical applicability. In our system, female expression cassette (1371) expressing plants generate complete male sterility when compared with control (pollen viability (%): 77.7 ± 1.3 (100%), pollen germination (%): 70 ± 3.6 (100%) and seed-setting (mg/pod): 74.23 ± 5 (100%)), 10 randomly selected BECLIN1 expressing transgenic lines showed pollen viability (%): 0.76 ± 0.78 (

0.96%), pollen germination (%): 0.76 ± 0.73 (

2.3%) and nil seed setting (Fig. 4u,v). The fertility restoration of F1 progeny works on transcription abolition of male sterility gene (BECLIN1) regulated through male parent. When COP1 expressing lines (1372) were used as male parent, F1 showed

21-fold reduction in BECLIN1 expression (Fig. 5c), which restored pollen viability (%): 15.14 ± 1.8 (20%), pollen germination (%): 18.5 ± 3.3 (26%) that is sufficient for optimal seed setting (mg/pod) 60.2 ± 15 (81%) (Fig. 4u,v). We observed further improvement in fertility restoration when COP1-mutant (COP1 L105A ) lines (1373) were taken as male parent, which completely abolished the expression of BECLIN1 resulting complete restoration of pollen fertility in F’1 with pollen viability (%): 74.58 ± 1.2 (96%), pollen germination (%): 69 ± 4.6 (

97.14% ) and seed setting (mg/pod) 71.9 ± 6.1 (97%) (Fig. 4u,v) comparable to the untransformed control plants.

Maintaining the male-sterile female lines is a prerequisite for future commercial application of this technique and the genetic design of the male-sterile female line (construct 1371, Fig. S1a-ii) provides this opportunity. Crossing the heterozygous male-sterile female parent (BECLIN1/−) with its wild type (−/−) results in

50% of the male-sterile progeny (1:1 ratio, (BECLIN1/−) and (−/−)). In future approaches, linking the herbicide resistance gene in the construct 1371 (as in SeedLink TM ) will enable the selection of male-sterile female parents. However, this requires overplanting and eliminating half of the sown plants by applying herbicide to obtain pure male-sterile female parents.

In conclusion, we have developed a system for tapetum-specific, high-level expression of the desired gene to achieve complete male-sterility, along with a system for transcriptional control over the expression system for fertility-restoration in the F1-hybrid. The tapetum-specific expression of the BECLIN1/ATG6 gene facilitated complete male sterility and COP1-mediated HFR1 degradation system was used for repression of transcription of BECLIN1 followed by fertility restoration in the F1 hybrid. The proposed male sterility-fertility restoration system described here will be a valuable future contribution for exploiting hybrid vigor and commercial production of hybrid seed.


Going to Seed with Dan Brisebois

Male Sterility in Plants

This post is an to answer Eric Weber‘s recent question about male sterility in seeds. Eric had been thinking about using Green Goliath broccoli in a breeding project but heard that High Mowing Seeds has been having a hard time producing Green Goliath seed and suspected male sterility. He wondered what caused this problem and what to do about it.

The disclaimer: This post is kind of heavy on genetics, that may or may not affect its reading enjoyment for some. Also, I am not on expert on this topic and might be inaccurate or downright wrong on some of my understanding – I don’t think I am that far off though.

Well, what is male sterility?

From Wikipedia: “Male sterility is the failure of plants to produce functional anthers, pollen, or male gametes.”

A male sterile plant cannot pollinate other plants or itself. However, the female reproduction structures of the flower are still fertile. A male sterile plant can therefore set seed if they are pollinated by a different (male fertile) plant. This genetic anomaly is used in hybrid seed production since it guarantees cross-pollination on male sterile plants.

There are 3 male sterile situations:

  1. Genetic Male Sterility
  2. Cytoplasmic Male Sterility
  3. Genetic Cytoplasmic Male Sterility.

1. Genetic Male Sterility

In this case, male sterility is caused by a recessive gene (ms). When both parents carry the recessive gene then the pollen is not viable . If you cross a genetical male sterile plant (msms) with a male fertile plant (MsMs), the F1 offspring will only carry one copy of the ms gene (Msms) and will be fertile. However, when you cross two of these F1 plants you will find the following genotyoes: MsMs, Msms, and msms. There will be some male sterile plants (msms) in addition to plants carrying the ms gene without expressing it (Msms).

I don’t know how easy it is to identify the male sterile plants in a large highly cross-pollinating population of a crop like broccoli. But if you can, and you rogue male sterile plants out, over many generations you will greatly reduce the frequency of the ms gene. This might be adequate to build a strong population.

If Green Goliath is genetical male sterile, Eric can probably use it in his breeding project as a mother plant. From what I’ve read though, genetic male sterility is not very common.

2. Cytoplasmic Male Sterility (CMS)

The cytoplasm is the stuff inside a cell wall in which the cell nucleus floats around in. Here’s a drawing. When a plant sets seed, some DNA from the mother plants cytoplasm is carried along. Cytoplasmic Male Sterility is transmitted by this type of DNA. If the mother was CMS, the offspring will be also.

This type of male sterility will shoot you in the foot if you are trying to breed an open pollinated population since there will be no gene flow from the CMS plants to any of the other plants.

3. Genetic Cytoplasmic Male Sterility

This is a CMS plant for which exist genes that can be introduced from another plant which will restore male sterility. This is great if you have plant breeding facilities and genetic material to work with but most of us farm scale breeders probably don’t have these resources.

If Green Goliath is either cytoplasmic male sterile or genetic cytoplasmic male sterile, it might be best for Erik to find some other broccoli variety to work with.

Well, if you know (or suspect) your variety is male sterility, then plant a row of the male sterile variety beside a row of a male fertile variety. The male sterile should set seed but all of the seed will be crossed with the male fertile.

Save the seed from each row separately,

The following year, only sow seed saved from the male sterile row.

If the plants don’t set seed, they are CMS. Your breeding project likely shuts down here.

If seed does set, the plant are probably genetic male sterile. The hard work begins now. The ms genes are still present in the population just waiting to be expressed. If you can identify male sterile plant in future generations, you can rogue them out than over time. Some head to row selection in 5-6 generations might help clean up this genetic nightmare.

Many hybrid seed is produced using male sterility. Especially onions, carrots, beets, and some brassicas. If you try to dehybridise such a variety, you might wind up with fertility problems sooner or later.


Hybrid Seed Production in Tomato

Botanical name: Solanum Lycopersicum L.

Family: Solanaceae

Chromosome no: 2n=24

Origin: Peruvian and Mexican region

Introduction

Hybrid tomato varieties have many advantages compared to open-pollinated varieties. Hybrids usually produce higher yields. They generally mature earlier and more uniformly. Many hybrids have better fruit quality and disease resistance. With all of these advantages, many farmers prefer to sow hybrid seeds in spite of the higher seed costs. The demand for hybrid tomato seeds can open a new market for growers interested in seed production. Hybrids have almost 5-7 times higher yields than open pollinated varieties. Hybrid tomato seed production is not simple. First, it is laborious process. Fortunately, this is not a problem in developing countries where affordable labor is available. Second, it requires the mastery of special skills and a close attention to detail. This publication will teach these skills.

For hybrid seed production we have to provide a certain temperature range for day and night periods as 15-20 Ċ and 20-28 Ċ respectively

Hybrid seed production involves the crossing of a female line to a male line. Either line can be the female or male parent, but normally the best seed yielder is selected as the female parent. Both parents should be pure, preferably being self-pollinated for more than six generations (this is called inbreeding). The inbred parents are selected for their desirable traits (e.g., high yields, disease resistance, fruit quality, earliness, etc.).

It is important to have plenty of pollen available for making hybrid crosses. Since tomato vines bloom profusely, a ratio of one male for every four female plants is recommended.

Self-pollination cannot be allowed in hybrid seed production. The female flower must be pollinated by the pollen from the male line. To prevent self-pollination, remove the stamens from the flower buds of the female line before they shed their pollen and this process is called emasculation.

Emasculation begins about 55-65 days after sowing. Flower buds from the second cluster which will open in two to three days are chosen for emasculation. Petals will be slightly out of the flower bud but not opened, and the corolla color is slightly yellow or even paler. Flowers from the first cluster are removed. Sterilize the forceps, scissors and hands by dipping them in 95% alcohol before emasculation is started. If gloves are used, these should also be dipped in 95% alcohol to prevent pollen contamination. Use sharp-pointed forceps to force open the selected buds. Then, split open the anther cone. Carefully pull the anther cone out of the bud, leaving the calyx, corolla and pistil .To help identify the hybrid fruits from self-fruits at the time of harvest, cut the corolla and calyx (all or two sepals)

Emasculation of Tomato: selection of buds, removal of anther cone, and cutting of petals

Collect flowers from the male parent to extract pollen. The best time for pollen collection is during the early morning before the pollen has been shed. Avoid pollen collection on rainy days.

Remove the anther cones from the flowers and put them in suitable containers, such as glassine, cellophane, or paper bags. Dry the anther cones by placing them 30 cm below a 100-watt lamp for 24 hours. The lamp creates a drying temperature of about 30°C. Pollen can also be sun-dried, but avoid drying at midday when temperature is very high. Put the dried anther cones in a plastic pan or cup.

Cover the cup with a fine mesh screen (200-300 mesh) and then seal it with a similar tight-fitting cup, serving as a lid. Shake the cup about 10-20 times so that the pollen is collected in the “lid” cup. Transfer the pollen into a small convenient-to handle container for pollination. Fresh pollen is best for good fruit-set. It can be kept for one day at moderate room temperature. When weather conditions are not suitable for pollination, dried or dehydrated pollen can be stored in a sealed container (capsule or vial) and kept in the freezer for about a month. Without freezing, the pollen can be kept in an ordinary refrigerator for two to three days without any significant loss in viability. The pollen should be taken from the freezer or refrigerator and kept closed until the container warms to room temperature. This will prevent the pollen from getting wet due to condensation.

Emasculated flowers are generally pollinated one to two days later. Try to avoid pollination on rainy days. The corolla of the emasculated flower turns bright yellow, signaling that the stigma is ready for pollination. Dip the stigma into the pool of pollen in the pollen container or pollinate by touching the stigma with the tip of the index finger dipped in the pollen pool Pollination is usually done three times weekly over a three to five week period. Successful pollinations are easily seen within one week by the enlargement of the fruit.

Pollination of Emasculated Flowers

The number of hybrid fruits produced per plant depends on the fruit size of the maternal parent. As a rule of thumb, maintain the following: 30 fruits for large-fruited parent 40 fruits for medium- fruited parent and 50 or more fruits for small-fruited parent. Hybrid fruits are easily recognized by their cut sepals. Remove the naturally-pollinated (non-hybrid) fruits, if any, from the female plants. This removal will prevent the accidental mixture of non-hybrid with hybrid fruits. Furthermore, non-hybrid fruits will steal nutrition away from the ripening hybrid fruit

Tomato fruits ripen about 50-60 days after pollination, but may take longer if temperatures are cool. Keep the fruits on the vine until they are fully mature, preferably to the pink or red ripe stage. This enables the seed to develop normally and fully. If fruits are harvested at an earlier stage, place them in a covered, cool dry place for three or four days until they become red ripe. Be sure to check for the clipped sepal before harvesting fruit. Collect fruits in nonmetallic containers, such as nylon net bags, plastic buckets, or crates. Metal containers may react with acids in the tomato juice and affect seed viability. Hence, they should not be used.

The work on tomato seed extraction and storage contains lot of care. For the storage of tomato seeds, the seeds were dried.

The fully ripped fruits like fully red tomatoes are selected for collection of seed and then their seed are extracted manually. For the extraction the tomatoes are placed in shopping bags by putting some water in it and remain it for one day. The next day seeds are extracted by shaking of shopping bags and washed with water. The seeds are dried in sun shine till 8 am to 1 pm and stored in packet. It’s a cheap and manual way of collecting seed. But it needs a lot of labor and it’s a time consuming task as well. Environment also affects this process, some time there is sun shine sometimes not.


Mass selection in cross-pollinated species takes the same form as in self-pollinated species i.e., a large number of superior appearing plants are selected and harvested in bulk and the seed used to produce the next generation. Mass selection has proved to be very effective in improving qualitative characters, and, applied over many generations, it is also capable of improving quantitative characters, including yield, despite the low heritability of such characters. Mass selection has long been a major method of breeding cross-pollinated species, especially in the economically less important species.

The outstanding example of the exploitation of hybrid vigour through the use of F1 hybrid varieties has been with corn (maize). The production of a hybrid corn variety involves three steps: (1) the selection of superior plants (2) selfing for several generations to produce a series of inbred lines, which although different from each other are each pure-breeding and highly uniform and (3) crossing selected inbred lines. During the inbreeding process the vigour of the lines decreases drastically, usually to less than half that of field-pollinated varieties. Vigour is restored, however, when any two unrelated inbred lines are crossed, and in some cases the F1 hybrids between inbred lines are much superior to open-pollinated varieties. An important consequence of the homozygosity of the inbred lines is that the hybrid between any two inbreds will always be the same. Once the inbreds that give the best hybrids have been identified, any desired amount of hybrid seed can be produced.

Pollination in corn (maize) is by wind, which blows pollen from the tassels to the styles (silks) that protrude from the tops of the ears. Thus controlled cross-pollination on a field scale can be accomplished economically by interplanting two or three rows of the seed parent inbred with one row of the pollinator inbred and detasselling the former before it sheds pollen. In practice most hybrid corn is produced from “double crosses,” in which four inbred lines are first crossed in pairs (A × B and C × D) and then the two F1 hybrids are crossed again (A × B) × (C × D). The double-cross procedure has the advantage that the commercial F1 seed is produced on the highly productive single cross A × B rather than on a poor-yielding inbred, thus reducing seed costs. In recent years cytoplasmic male sterility, described earlier, has been used to eliminate detasselling of the seed parent, thus providing further economies in producing hybrid seed.

Much of the hybrid vigour exhibited by F1 hybrid varieties is lost in the next generation. Consequently, seed from hybrid varieties is not used for planting stock but the farmer purchases new seed each year from seed companies.

Perhaps no other development in the biological sciences has had greater impact on increasing the quantity of food supplies available to the world’s population than has the development of hybrid corn (maize). Hybrid varieties in other crops, made possible through the use of male sterility, have also been dramatically successful and it seems likely that use of hybrid varieties will continue to expand in the future.


Discussion

Hybrid seed production and utilization has revolutionized the crop production by exploiting the genetic phenomenon of heterosis/hybrid vigor. This technological advancement directly contributed to an increase in the yield as seen in rice where hybrid rice yield increased

10–20% over the conventional lines [23]. There is a high potential to exploit the heterosis in soybean hence, understanding molecular basis of male sterility in soybean mutant lines is critical for the development of high yielding hybrid varieties. Further, insects have shown to be involved in transferring pollen from male-fertile plants to male-sterile soybean plants [24] which avoids laborious manual pollination. Identification and characterization of soybean male-sterile lines are critical for producing hybrid seed at commercial level as well as to understand the mechanism responsible for male sterility. In addition, the genetic male sterility has an advantage over the cytoplasmic male sterility (CMS) that it does not require a dedicated maintainer line to propagate the male-sterile line. The major constraint in the utilization of the genetic male sterility for production of pure hybrid seeds is the identification and removal of the male-fertile plants before flowering. Successful pre-flowering recognition of homozygous male sterile plants requires specific morphological or molecular markers. Male-sterile lines often do not display visible phenotype before flowering hence the gene-specific molecular markers are more practical in the identification and removal of male-fertile plants. In addition, these markers can be used for male-sterile line selection in backcrossing and recurrent selection breeding programs [3]. Further, identification of photoperiod/thermo-sensitive genic male sterile (PTGMS) lines may provide flexibility in regulating fertility by managing environmental conditions [25]. Hence, developing independent male-sterile lines and understanding the molecular mechanisms will help to deploying a suitable approach for hybrid seed production. In soybean, eleven independent male-sterile lines (ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, msMOS, and msp) generated by various methods (spontaneous, fast neutron irradiation and transposable elements) have been identified [6,7,8]. In addition, the availability of the male-sterile lines from diverse genetic backgrounds further facilitates the opportunity for the exploitation of genetic diversity from different lines. Despite the identification of several male-sterile lines in soybean, not much is known about the identity of the genes or molecular mechanism behind the male sterility.

The overarching goal of the present study was to identify and characterize the gene responsible for male-sterile phenotype of the ms4 mutant. The ms4 was previously mapped to a 694 kb region on chromosome 2 which harbored 88 genes [6]. The soybean msp gene was also mapped in the same region hence fine mapping to identify the actual causal gene was essential to develop gene-specific molecular markers [6]. It is believed that ms4 is different from msp, as the mechanisms involved in governing male sterility are different [6] indicating that mutations in independent loci are responsible for the male sterility in these lines. The msp is a temperature sensitive male sterile mutant, which displays higher fertility in the hot environment compared to cooler temperatures [14], while ms4 is not a temperature sensitive mutant. In the present study, we have fine mapped the ms4 locus to

216 kb region that contained 23 protein-coding genes (Fig. 2 Additional File 1: Table S1) and candidate gene was identified based on the functional relevance. The candidate gene (Glyma.02G243200) displayed high homology to Arabidopsis MMD1 which is involved in the male fertility in Arabidopsis [16,17,18]. The PHD domain protein AtMMD1 has been shown to be essential for proper chromosome condensation during male meiosis [16,17,18]. The mmd1 mutant fails to produce viable pollen due to pollen degeneration after the tetrad stage, a phenomenon that is also observed in the ms4 mutants, endorsing Glyma.02G243200 as the candidate gene. Sequence analysis of Glyma.02G243200 from ms4/ms4 plants confirmed the presence of a spontaneous mutation resulting into a truncated protein lacking PHD domain (Fig. 3B Additional File 1: Figure S1 and S2) [17, 18]. The PHD domain has been shown to be critical for MMD1 function in Arabidopsis [16, 18] which is missing in ms4/ms4 plants due to spontaneous mutation resulting in truncated protein.

Interestingly, phylogenetic analysis of the MS4 protein revealed the presence of another PHD domain protein, MS4_homolog, encoded by Glyma.14G212300 gene on chromosome 14. These two homologs appear to be result of segmental duplication during paleoploidization event (Additional File 1: Table S3). Even though cytogenetically soybean behaves as diploid, there are reports of at least two rounds of whole genome duplication [26, 27]. It is interesting that despite the presence of a homolog, the Ms4 is the only gene that is governing the male-sterile phenotype. These results are supported by the expression data which shows that soybean Ms4 expression is significantly higher than Ms4_homolog in most of the tissues, except root (Fig. 5). There are several reports of similar expressional shift (spatial and quantitative) among the remained duplicated genes (paralogs) in plants including soybean [28]. Non-functionality of MS4_homolog can also be explained based on the study in which loss or silencing of approximately 25% of the duplicated genes in soybean were reported since the last duplication event [26, 28]. The Arabidopsis homozygous mutant lines (mmd1/mmd1) complemented with soybean Ms4 (AtMMD1pro::gMs4 and AtMMD1pro::cMs4) showed successful functional complementation by producing viable pollen and siliques with seeds, whereas the Ms4_homolog was not able to complement (Fig. 6). Since, both Ms4 and Ms4_homolog genes were driven by native MMD1 promoter, the functional characterization clearly showed lack of function for Ms4_homolog, which explains the reason behind the male sterile phenotype of ms4 despite the presence of another homolog. The failure of Ms4_homolog to rescue the male-fertile phenotype could be attributed to the 7% differences in amino acid composition between these two proteins (Additional File 1: Figure. S4). Further studies are needed to understand the reasons for the functional differences. In addition to its inability to produce viable pollens, homozygous mmd1 mutants produce shorter filaments placing the anthers below the stigma (Fig. 6). The complemented Ms4 lines produced normal filaments and viable pollens suggesting that the PHD finger proteins are involved in the filament elongation in addition to the chromosomal condensation (Fig. 6). Overall, our data successfully demonstrated that the spontaneous mutation in Glyma.02G243200 that resulted in premature stop codon is responsible for the male-sterile phenotype of the soybean ms4 line.


DISCUSSION

Pollen production is essential for pollination, which is the first step in setting seed. Although failure to pollinate (male sterility) can prevent seed set in self-pollinated plant species, male sterility is highly beneficial in hybrid breeding. The discovery of male sterility traits in plants has enabled breeders to produce hybrid seeds much more efficiently in a wide range of crops ( Kim and Zhang, 2018 ). The direct benefit of hybrid production has been demonstrated by the significant yield advantage of maize hybrids over land races as well as improved yields and fitness in many other major crops and bioenergy species ( Zhang et al., 2018 ). Control of pollen production is critical for hybrid breeding especially for hybrid seed production in self-pollinated species. Therefore, identifying male sterility in important crop species and improving their use in hybrid breeding systems could make important contributions to increasing future agricultural production and food security.

No gene that mediates NMS in sorghum has been cloned previously despite the discovery of several sorghum NMS lines and mutants ( Andrews and Webster, 1971 Pedersen and Toy, 2001 Xin et al., 2017 ). Here, we report the isolation and characterization of a new male-sterile mutant and the identification of the first NMS gene in sorghum. This mutant, designated ms9, is distinct from all other sorghum NMS lines reported previously ( Andrews and Webster, 1971 Pedersen and Toy, 2001 Xin et al., 2017 ). The male-sterile phenotype in ms9 mutants can be easily recognized at onset of anthesis because of its thin pale anthers and exaggerated stigmas. Other than male sterility, the ms9 mutants develop similarly to WT BTx623 plants. The characteristics of ms9 make it ideal for development of a two-line breeding system in sorghum based on NMS ( Chang et al., 2016 Zhang et al., 2018 ).

The identification of Ms9 as the causal mutation for the male-sterile phenotype of the ms9 mutant is supported by bioinformatic analysis of two independent whole-genome sequencing data sets of pooled F2 mutants as well as by identification of another independent allele (Fig. 5) from the sequenced mutant library ( Jiao et al., 2016 ). The cloned Ms9 gene encodes a PHD-finger transcription factor with a gene structure very similar to that of Ms1 in Arabidopsis, Ptc1 in rice, and Ms7 in maize ( Li et al., 2011 Wilson et al., 2001 Zhang et al., 2018 ) as well as homologs in other cereals and plant species (Fig. 5C). The mutations identified in the two ms9 mutant alleles (R218W and A37V) cause amino-acid changes in the conserved domains of this protein (Fig. 6). The amino-acid sequence of SbMS9 is also very similar to its orthologous counterpart other crops (Supplemental Fig. S2). MS7, recently identified in maize, is the closest homolog to SbMS9, with 100% identity in the conserved region (Fig. 5C, 6). The phenotype of ms9 is similar to that of the male-sterility phenotypes of Arabidopsis ms1, the rice ptc1 and maize ms7 lines ( Li et al., 2011 Wilson et al., 2001 : Zhang et al., 2018 ), all of which have significant effects on anther morphology and pollen development but no effect on other aspects of floral development and morphology (Fig. 1, 3, 4).

The expression pattern of the Ms9 gene is also similar to its orthologs in other species. The expression data of Ms9 gene was extracted from the Morokoshi Sorghum Gene Expression Atlas ( Makita et al., 2015 ). SbMs9 is highly expressed in young inflorescence tissues and anthers (Supplemental Fig. S3A). This tissue-specific gene expression pattern is similar to that of Ms1 in Arabidopsis and Ptc1 in rice ( Li et al., 2011 Wilson et al., 2001 ), further supporting the role of Ms9 in the development of tapetum and pollen grains. As with Ms1 and Ptc1 in Arabidopsis and rice, Ms9 may serve as a critical regulator in tapetal cell degeneration and pollen development during anther development.

At present, the three-line hybrid breeding system relies exclusively on CMS for the male-sterile female parent in sorghum ( Praveen et al., 2015 Rooney, 2004 ). Although several types of cytoplasmic male-sterile lines are available, commercial hybrid production uses mainly the A1 cytoplasm ( Jordan et al., 2011 ). A main advantage of the three-line breeding system in sorghum is that good A–B pairs can produce nearly 100% male-sterile line to serve as a female parent during production of hybrid seeds. Furthermore, this system has been used in breeding grain sorghum since 1940s, and many breeding materials in grain sorghum have been converted for use in this breeding system ( Stephens and Holland, 1954 ). However, several aspects of the CMS breeding system need to be improved. For example, many sorghum accessions are neither perfect B nor R lines and cannot be used in breeding sorghum hybrids without lengthy period of conversion to B or R lines. In addition, many lines suitable for breeding biomass or sweet sorghum hybrids have not been converted and cannot be used directly to breed hybrids with the CMS-based breeding system. Furthermore, The A1 cytoplasmic homogeneity may predispose sorghum hybrids to devastating diseases, as in the T-cytoplasmic maize hybrids produced in the 1970s ( Ullstrup, 1972 ).

A two-line breeding system that uses NMS can potentially overcome the disadvantage of the CMS-based three-line breeding system. For example, the male-sterility of a two-line breeding system based on a nuclear mutation can be restored by any line that does not carry the same mutation ( Chang et al., 2016 ). This advantage is particularly useful for breeding biomass and sweet sorghum hybrids because accessions with the useful bioenergy traits can be directly used as parents for the hybrids. The two-line breeding system based on NMS also does not require male-sterile cytoplasm and, therefore, can avoid homogeneity of cytoplasm in hybrid. The main disadvantage of directly using NMS in hybrid breeding is that a fertile plant, heterozygous for an NMS mutation, only produces 25% of homozygous male-sterile plants. To remove the fertile plants from a breeder's field is nearly impossible. Fortunately, through strategic manipulation of the NMS gene and its mutation, the two-line breeding system has been shown to produce pure male-sterile lines in rice ( Chang et al., 2016 Huang et al., 2014 Zhou et al., 2014 ). In Maize, Ms7, the closest homolog of the sorghum Ms9 gene, was identified by map-based cloning and used, along with its WT gene, to engineer a controllable male-sterile line for a two-line breeding system ( Zhang et al., 2018 ). A gene construct with the WT Ms7 gene and multiple control elements has been used to rescue the male sterility of the ms7 mutant and produce pure male-sterile seeds ( Zhang et al., 2018 ). The identification of the Ms9 gene and its causal mutations provides critical tools to manipulate the production of male-sterile parent that is 100% male sterile.

In summary, we characterized a new sorghum NMS mutant, ms9, and identified the first NMS gene in sorghum. The identification of the SbMs9 provides an opportunity to engineer controllable male sterility for development of a two-line breeding system in sorghum.

Supplemental Information Available

Supplemental information is available with the online version of this manuscript.

Conflict of Interest

The authors declare that there is no conflict of interest.

Author Contributions

JC and ZX conceived the idea and designed experiment, YJ analyzed the data, HL performed histological and SEM analyses of anther features, all performed the experiment. JC and ZX drafted the manuscript with input from all authors. All authors agree with the final manuscript.

Disclaimer

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity provider and employer.


Watch the video: Male sterility. 3 line system of Hybrid seed production for JRF by Tanisha Gangrade (September 2022).


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