Principles of Variety Maintenance for Quality Seed Production

Abstract

Development of new plant varieties is key to sustainable agriculture, specially given that climate change poses newer challenges with uncertainties of production ecosystems. However, to accrue the full genetic potential of a variety, it is essential to maintain the variety in true-to-type, as it was released for commercial use. The methodology adopted to maintain the genotypic constitution of a variety through the series of multiplication (generations) is known as variety maintenance or maintenance breeding. Its successful implementation needs a thorough understanding of the breeding methodology, varietal characteristics, and the influence of the environment on them. Though specific procedures of variety maintenance are followed for each crop group, which are based on their flowering, pollination behaviour and other essential traits, the basic principles are based on the mode of pollination (self- or cross-pollinated) and genetic constitutions.

1 Introduction

Productivity of a variety is the result of improved genetics (genetic gain) coupled with better crop management. Variety development and seed production are complementary activities and one without the other has little relevance in the context of agriculture. To realize the genetic potential of the new and improved varieties, a strong seed multiplication must be linked to their distribution, and marketing system (Seth et al. 2009). Significant strides in productivity in major cereals have been made in India since the 1950s, specially with the ushering of the Green Revolution, as the combined outcome of the enhanced genetic potential of improved varieties, availability of genetically pure seeds of such varieties, and improved crop management practices (Yadav et al. 2019).

Seed security is a prerequisite for food security, and this is amply demonstrated by the linear increase in grain production and availability of genetically pure seeds (Indian Seed Statistics: Perspectives 2019), thus, crop improvement is the key to a successful seed programme. Plant breeding per se is man-directed ‘plant evolution’ and one of the chief limitations of traditional breeding is that selection decisions are primarily based on phenotypes. The phenotype is the manifested expression of one or more traits, whereas genotype reflects the allelic composition of an individual at one or more loci (Singh and Singh 2017). The different traits of an individual can be categorized into two groups: (i) qualitative traits which are governed by one or few major genes/oligogenes, each of which produces a large effect on the characteristic phenotype, and (ii) quantitative traits which are controlled by many genes, each having a small effect on a characteristic phenotype, and are generally cumulative. From a breeding perspective, most of the traits of interest are metric or quantitative in nature, whose phenotypic expression is significantly impacted by environment and also genotype and environment interactions.

For a practising maintenance breeder, the following equation best expresses the phenotype and its constituents:

$$ P=\mu +G+E+\left(G\times E\right) $$

where P is the phenotype of a quantitative trait (controlled by multiple genes), μ is the population means, G is the genotype effect of the individual concerned, E is the environmental effect on trait expression and (G × E) is the interaction component. An accurate assessment of G, E and G × E components of phenotypic variation for different quantitative traits is one of the perpetual pursuits for plant breeding (Singh 2012). This assessment and judgement about G, E and G × E also pose a dilemma in maintenance breeding, as the production of different classes of seeds, including the multiplication of nucleus and breeder seed, is commonly based on the phenotype, except in few cases where laboratory analysis is required to ascertain a trait.

1.1 Quality Control: An Essential Prerequisite of Varietal Maintenance and Seed Multiplication

The role of quality control in varietal maintenance and seed multiplication is of paramount importance for maintaining delineated traits and making them available to end-users in their exactitude. In seed multiplication, a generation system is followed to produce an adequate quantity of seed from a small quantity of the purest seed available, and a statutory system is devised for field inspection and approval of the scheme leading to seed certification. All certification systems viz., OECD schemes, AOSA and Indian seed certification systems are essentially based on the same philosophy of generation system, and similar procedures. Variety purity standards are maintained by using a set of harmonized procedures during seed production. The control scheme for varietal maintenance and seed multiplication per se follows a generation system with seed class denominations typical for the specific certification system. The case study in wheat depicts the systematic augmentation of seed quantities and multiplication area involved to increase the seed from the generation of ‘nucleus’ or ‘breeder seed’ (Table 1) from 0.4 t up to 64,000 t for the generation of the “truthfully labelled” or “commercial stage two” class seed (TL or C2) indicating the need of a robust system with varietal purity as central tenet at each multiplication seed class (Table 2).

Table 1 Generation system in wheat followed for systematic seed multiplication in India vis-a-vis multiplication stages in OECD with emphasis on seed multiplication ratio

Table 2 Analogy of field inspection in Indian seed certification and EU system of seed certification

2 Maintenance Breeding

The term maintenance breeding, often used interchangeably with varietal maintenance, is the foundation to a quality seed production programme. Without proper maintenance breeding, varieties deteriorate rapidly in terms of their genetic potential, and lose much of their value for cultivation, irrespective of their performance traits at the time of release. Maintenance breeding, therefore, is the key to the purification and stabilization of released varieties and varieties to be released, which help to disseminate and enhance the productive life of a variety. Combining the art and science of Plant Breeding with applied aspects of seed production is needed for the maintenance of plant varieties. It is based on the fundamental principles of plant breeding, but the real task during seed multiplication is to identify both obvious and cryptic variants and eliminate these in the initial generations of purification so that only the true-to-variety material is scaled up in subsequent seed multiplication stages. With wheat as a case study, screening for phenotypical off-types needs many visits through the fields within the growth stages (Fig. 1) of ear emergence and dough stage. The intense inspections have to be in the initial multiplication classes, irrespective of the national or international certification scheme. For example in wheat, many breeders and seed multiplication services recommend 8 inspection visits through the fields of “nucleus seed” production, while reducing their number successively to two in fields of “certified seed” production (Weissmann 2022, unpublished data).

Fig. 1

Time span (blue arrow) and number of field inspections (visits) to check uniformity and purity during maintenance and multiplication in a wheat crop (Weissmann 2022)

2.1 Objectives of the Maintenance Breeding

The basic tenets of varietal maintenance, on which a robust seed multiplication programme relies, are as follows:

• Systematic multiplication of seeds (hastening up through generation system).

• Increase the homozygosity, which in turn can lead to an increase in the homogeneity.

• Elimination of off-types thereby maintaining the stability.

Maintaining the stability depends on contrivances for the occurrence of off-types viz. unintentional mixing during seed multiplication, genetic segregation at individual loci, pollination from other plants in the neighbourhood (1–3%) and natural mutation (with a frequency of 1 × 10⁻⁶). Hence, breeders involved in varietal maintenance should have a thorough understanding of breeding behaviour and the impact of environmental conditions, varietal descriptors of the variety/parental lines (of hybrids) and specific requirements like isolation, land requirements together with the impact of the pressure of biotic factors.

During different stages of seed multiplication, there may be contamination and even complete loss of certain desirable trait (s). Hence, the prevention of contamination gets top priority in variety maintenance programmes (Priyadarshan 2019). This requires a comprehensive knowledge of the flowering and pollination behaviour of the respective species, typical morphological characteristics of the variety as per DUS testing, and the ability to identify possible variation(s) due to growing conditions. For example, there could be multiple GxE interactions during seed production influenced by the micro-environments, which may trigger the expression of various off-types. Differentiating the true variants from the temporal variants due to growing conditions poses a big challenge to the seed production professionals during the maintenance breeding. Therefore, it is desirable to take up the maintenance breeding programme of the variety in its area of adaptation following the GAP recommended for the crop.

When maintenance breeding is undertaken carefully, by combining field observation for morphological characters and some laboratory tests for quality traits viz. organoleptic and cooking quality; fatty acid profiles; protein profiles (gliadins for bread making purpose) and/or useful nutrient(s) for which the variety has been bred; it results in quality seeds, desired agronomic performance, and hence, a longer life in cultivation.

2.2 Methodology

The maintenance breeding would be a function of any of the four fundamental breeding schemes (Table 3), as described by Simmonds and Smartt (2014). Hence, the practices adopted for varietal maintenance of different crops primarily depend upon the mode of reproduction (asexual or sexual) and the mode of pollination (self or cross or often cross-pollination). These reproductive modes/mating systems are responsible for the kind of variants/off-types, which might be expected and observed in a seed production programme.

Table 3 Fundamental populations in plant breeding (based on Simmonds and Smartt (2014))

A sound understanding of the mating system of the crop is a prerequisite for undertaking an appropriate maintenance breeding programme (Yadava et al. 2022). In self-pollinated crops, like wheat and rice, nucleus seed is produced by growing plant-to-rows, i.e. evaluating selected true-to-the-type plants on the basis of performance of their progenies. The plant-to-row method is suitably modified, depending upon the growth habit of the crop plants, to panicle-to-rows in rice, ear-to-rows in wheat or cluster rows in cowpea (Yadav et al. 2003). Plant-to-row method is used as such, in crops like green gram, chickpea, field pea and lentil, where single plants can be taken out easily. In cross-pollinated and often cross-pollinated crops like maize, pearl millet, pigeon pea and mustard, the plant-to-row methodology is not very effective, as it does not exclude genetic contamination through pollen of off-type plants. The method used in such crops is Reserve Seed Method. In this method, single plants typical of the variety are selected, individually harvested, threshed and screened for seed traits. In the next year, only a small part of the seed of each selected plant is planted as plant rows; the remaining seed is stored as reserve. The plant rows are carefully screened before and after flowering and until harvest. True-to-type uniform plant rows are identified. The reserve seed from the plants that produced true-to-type progenies is bulked and used as the nucleus seed.

2.2.1 Self-Pollinated Crops

2.2.1.1 Rice

Most of the seed production systems in the world follow 3 or 4 generations of seed multiplication starting from pre-basic or breeder seed. In India, there are three acknowledged classes of seed, i.e. breeder (BS), foundation (FS) and certified (CS). The seed chain follows a three or four-tier system of multiplication (BS → FS → FS/CS → CS). The initiation of seed multiplication chain is from breeder seed (a product of highest quality nucleus seed). If the breeder seed is not of high genetic purity, contaminants present get multiplied exponentially in the succeeding generations. This may result in complete erosion of the identity and loss of desirable attributes of a variety. Avoidance of contamination and prevention of genetic deterioration are therefore essential prerequisites of any effective seed programme. Varietal maintenance of basmati and non-basmati rice varieties for decades at the Regional Station of the Indian Agricultural Research Institute, Karnal is a leading example of the efficacy of this approach in enhancing the longevity of these varieties.

Rice varieties Pusa Basmati 1 and Pusa 44 were notified in 1989 and 1994, respectively, but the demand for the breeder seed of these varieties is still very high. This is made possible only because of the appropriate and effective maintenance breeding by the concerned institutions/researcher(s). The best approach for varietal purification, maintenance, and nucleus seed production of rice is the Panicle- to-row method (Figs. 2 and 3) and outlined below:

• Around 350 to 500 true-to-type single panicles are selected.

• Selected panicles are threshed individually and are thoroughly examined for seed characteristics (seed length, width and shape, etc.).

• In the case of basmati varieties, a portion of the seed is also tested for cooking analysis (kernel elongation, aroma, etc.).

• In the case of molecular marker-assisted backcross breeding (MABB) varieties, incorporated genes are also screened for their presence (e.g., Pusa Basmati 1718 possessing bacterial blight (BB) resistance genes namely xa13 and Xa21) or another basmati variety Pusa Basmati 1847 having two genes (xa13 and Xa21) for BB resistance and two genes (Pi54 and Pi2) for blast resistance).

• Seeds of panicles not matching to defined seed characteristics or not meeting the defined cooking quality benchmarks or any single panicle having an accidental plant without having the R-allele of the disease resistance gene are straightway rejected.

• Seeds of remaining (about 200–250) panicles are grown in panicle-to-rows or paired rows (from a single panicle).

• Thorough examination of panicle rows is done for their standard diagnostic traits at different crop growth stages.

• Panicle rows not matching to the typical plant type of the variety are completely removed.

• The remaining selected panicle rows are harvested and threshed individually and the seed of each panicle row is critically examined.

• Eventually, the seed of the selected true-to-the-type panicle rows is bulked to get genetically pure highest quality nucleus seed.

Fig. 2

Systematic procedure and ideal stage of initiating maintenance breeding in any rice varietal development and seed production programme (Atwal et al. 2009)

Fig. 3

Varietal maintenance plots (Panicle-to rows) of Pusa Basmati 1121 at ICAR IARI, Regional Station, Karnal

The incorporation of cooking tests (Fig. 4) and scrutiny for disease resistance genes using molecular markers in the varietal maintenance programme has significantly improved the market acceptability of these varieties. The emphasis is on stable diagnostic traits, cooking and quality characteristics (kernel length, elongation ratio, aroma) along with screening for disease resistance genes (MABB varieties), wherever applicable.

Fig. 4

Cooking Test showing kernel elongation: An integral part of varietal maintenance of Basmati rice

It is to be noted that roguing (in terms of taking out off types) is never undertaken in the varietal maintenance plots (nucleus seed plots). It is the straightway rejection of panicle rows expressing any sort of variants. Roguing operations are undertaken only in large-scale seed production plots (breeder, foundation, certified and truthfully labelled seed plots) (Seth et al. 2022).

2.2.1.1.1 Pusa Basmati 1121

A superb basmati rice variety with remarkable grain and cooking quality was notified for commercial cultivation in 2005. The superior linear cooked kernel elongation of this unique variety was derived from its parents Basmati 370 and Type 3. Amassing of favourable loci for extra-long grain and exceptionally high linear cooked kernel elongation was because of transgressive segregation due to selective inter-mating of the sister lines showing better linear kernel elongation in the segregating generations. A total of 13 rice varieties/enhanced germplasm including Basmati 370 and Type 3 were used to bring together the favourable alleles at multiple loci for grain and cooking quality characteristics and agronomic advantage in the development of Pusa Basmati 1121 (Singh et al. 2018). These novel varieties are the outcome of multiple crosses and intricate pedigree (Fig. 5), thereby making the varietal maintenance of these a highly specialized task. A range of off-types show up in the repeated cycles of seed multiplication of these varieties, and often it becomes very challenging to maintain the precise combination of favourable alleles of the specific variety during the repeated cycles of seed production.

Fig. 5

Pusa Basmati 1121 pedigree showing the contribution of several varieties. Years in parentheses indicate the year in which crossing was initiated and release of the variety, after Singh et al. 2018

Pusa Basmati 1121, a variety in great demand, is a distinctive example of the importance of variety maintenance, in a variety having a complex parentage, throwing different types of variants such as (i) Tall and dwarf off-types (Fig. 6); (ii) Grain size variants and (iii) Long awned off-types (Seth et al. 2022). Repeated cycles of varietal maintenance have enabled this variety to retain its predominance among Basmati rice varieties both in the domestic and international markets, making a significant contribution to farmers’ prosperity.

Fig. 6

Pusa Basmati 1121 purification and maintenance. Distinct off- types in a paired row, raised from single true to type panicle (Seth et al. 2022)

2.2.1.1.2 Pusa 44

A non-basmati variety is popular in northern India (Joshi et al. 2018, Dwiwedi et al. 2021), due to its high yield (8–10 t/ha) and suitability for mechanical harvesting. It is a semi-dwarf indica rice variety, with a sturdy culm, with long slender grains and high head rice recovery, and has a significant share in domestic consumption as well as rice export (Indian Seed Statistics (2019). Unlike Pusa Basmati 1121, Pusa 44 is a very stable variety to maintain. Only a few variants, mainly grain size, are observed in the Nucleus/Breeder seed plots. The popularity of this variety has grown manifolds since its release due to sustained varietal maintenance and availability of quality seed.

The above case studies amply showed the critical role played by varietal maintenance in any effective seed multiplication programme. With every step in the generation system of seed multiplication, the ratio of the volume of seed increases significantly from one generation to the next, depending upon the seed multiplication ratio of the crop. Maintaining varietal purity by examining every plant in large plots of Breeder, Foundation and Certified seed, is neither practicable nor cost-effective. Hence, utmost care should be taken at the initial nucleus seed production (Mandal et al. 2010).

2.2.1.2 Wheat

The variety maintenance in wheat presents a typical case of a self-pollinated species. A seed sample of a particular variety obtained from the concerned breeder is raised in isolation and around 500 spikes or ear-heads which are true-to-type (based on DUS characteristics), are selected and threshed separately. These single-ear seeds are examined for distinct varietal traits and those not conforming to the variety are rejected, and seeds from the true-to-type ear-heads, which did not show any variants upon table examination, will be stored separately for the subsequent stage of nucleus seed production (Table 4).

Table 4 Seed multiplication and maintenance breeding in wheat

2.2.1.2.1 Nucleus Seed Stage I (NSS1)

Seeds from selected ear-heads (G1) are planted in rows of 3 m length with isolation distance of 5 m from other varieties. Preference should be given to planting of nucleus seed plot surrounded by breeder seed crop of the same variety on all the sides to prevent any chance of out-crossing (Fig. 7). NSS1 seed is usually sown in single rows or may be in paired rows for sake of ease in the inspection. With any sort of deviation from delineated varietal descriptors, the entire row is discarded. If a genetic variation is detected in the progeny at or after the flowering stage, reject the progenies on both sides of the deviant progeny to prevent variation due to natural out-crossing in subsequent progenies, if the variation appears obviously to be heritable in nature. Progeny from each ear-head is to be harvested and threshed separately for multiplication in the next generation.

Fig. 7

Nucleus seed production of wheat at ICAR-IARI, Regional Station, Karnal. (Ear-rows of varieties HD 2428 or HD 2967 NSSII surrounded by breeder seed of the same varieties)

2.2.1.2.2 Nucleus Seed Stage II (NSS2)

If the need for source seed for breeder seed production is high, NSS2 is undertaken. In this method, the seed produced from each ear row of NSS1 is grown separately in larger plots. The number of such plots may vary depending upon the requirement of Breeder seed (BS). The plots are examined for any variation and variant plots are rejected as and when observed. True-to-the-type plots are harvested and threshed in bulk. This constitutes NSS2 which is usually sufficient enough to produce the desired volume of BS.

Maintenance of Hybrids in a Self-pollinated spp., e.g., Rice, Wheat(EU).

• Hybrids are developed using CMS system by involving three lines (Table 5) viz.

• A or CMS line (male sterile);

• B or maintainer line (male fertile) and

• R or restorer line (male fertile).

Table 5 Maintenance breeding and seed production of hybrids in self-pollinated crops

The success of hybrid seed production programme depends on the purity of parental lines. Maintenance and multiplication of the A-line are done by crossing it with B-line (A×B). Genetic purity of parental lines is the most important prerequisite to ensure the purity of hybrids. Maintenance of the A-line can also be achieved by using plastic barriers (Fig. 8). Since B and R lines are fertile, their maintenance and nucleus seed production are similar to that of self-pollinating inbred varieties.

Fig. 8

Line maintenance (ear-to-row) isolation with plastic walls, in hybrid wheat, Germany (Weissmann 2022, unpublished)

2.2.2 Often Cross-Pollinated Crops, e.g., Pigeon Pea

Being an often cross-pollinated species, maintaining a proper isolation distance, field inspections just before and at the time of flowering, and roguing are essential aspects of varietal maintenance in pigeon pea. Pigeon pea varieties may be bred as OPVs or hybrids.

2.2.2.1 Nucleus Seed Production of Varieties and/or Restorer Lines of Hybrids

Initially, 500–1000 true-to-type plants are selected from the basic/foundation seed plot, which is in isolation of 250 m from the plots of other varieties. Selected plants are bagged for selfing before the onset of flowering. Selfed seeds are harvested separately and are examined for distinct varietal descriptors. Non-conforming individual plant progenies are rejected and selected individual plant progenies raised in plots of 4 m row length with a spacing of 60–75 cm (row to row) keeping an isolation distance of 5 m between the progeny rows. Plant progeny rows showing variation should be completely rejected. Uniform progeny rows are harvested separately and examined for seed size, shape, and other distinct characteristics. Uniform progenies are bulked to constitute nucleus seed. In case, there is a higher requirement of nucleus seed, one more cycle of multiplication may be repeated.

If the variety is newly released and still segregating or needs purification then this method may not be very effective. In such case, we should go for reserve-seed method for purification of the variety, as described below:

• 1. Select a large number of single typical plants of the variety from base population raised from seed supplied by the breeder or from nucleus seed plot.

• 2. Harvest and thresh the selected single plants individually and examine for seed traits. Variant seed packets are rejected. Seed of the so retained seed packets is divided into two parts maintaining their identity.

• 3. One part is used for sowing in plant rows for evaluation in the next year, while the remaining part is stored as reserve seed maintaining its identity.

• 4. The single plant progeny rows are screened critically at regular intervals. A row with variant plant(s) is rejected.

• 5. During flowering the nucleus seed crop is examined daily or at alternate days to avoid cross-contamination. The rejected plant rows should be uprooted and removed from the field immediately after detection.

• 6. The retained rows are harvested and threshed separately and each separate seed lot is again examined for seed characteristics.

• 7. The reserved seed of single plants which produced uniform progenies typical of the variety is bulked to constitute the nucleus seed. The pure nucleus seed so produced is used for further multiplication or breeder seed production.

2.2.2.2 Nucleus Seed Production of A Line of Pigeon Pea Hybrids

In case of the cytoplasmic-genic-male sterility (CGMS) system, A and B lines are planted in a ratio of 4:1 with an isolation distance of 250 m. Any off-type plant should be rogued out before flowering. Conforming male sterile plants are harvested and threshed separately and after examining the seed characteristics, these are bulked as the nucleus seed of A line, whereas regarding B line, plants with designated varietal descriptors are separately harvested.

2.2.3 Cross-Pollinated Crops, e.g. Sunflower

2.2.3.1 Nucleus Seed Production of Open-Pollinated Varieties

About 5000 plants are selected in the base population which are raised under isolation. Selected plants are evaluated for varietal descriptors, of which 25% plants are advanced for progeny testing, these plants are raised in single progeny rows and for every 10 to 20 progeny rows a check cultivar is included for evaluation purpose. From each plant-progeny-row, a portion of selfed seed is retained as reserve seed. After harvest of progeny trial, laboratory evaluation is conducted to earmark superior progenies and reserve seed of superior progenies which was saved from individual plants is bulked to form nucleus seed (Fig. 9).

Fig. 9

Nucleus seed production of sunflower at the University of Agricultural Sciences, Bengaluru. Maintenance of A & B Lines in the CGMS System

A and B lines obtained from the original populations are used for sowing in ratio 1B: 2A:1B of 4 m length with about 300 rows of each line. Plants conforming to all the varietal descriptors are tagged and the pollen is transferred manually from B line to A line (paired crossing), while in B line selfing is done. Adept bagging and tagging are enabled for selected capitula in about 400–500 A × B crosses. Selected B lines are harvested first and evaluated for seed varietal descriptors. A lines are harvested later than the corresponding B lines from which they received pollen and threshed separately. After laboratory examination, conformed seeds from selected plants are taken in progeny rows. Both A and B line progeny rows not conforming to varietal descriptors are rejected. Individual A line plants are observed for pollen shedders. If any pollen shedder is found the whole line and its corresponding B line are rejected. Reserve seeds from conformed progenies of both the lines, after bulking separately form the nucleus seed respectively (Nucleus and breeder seed production manual, DSR, Mau 2010).

2.2.3.2 Nucleus Seed Production of R Line

Seeds from original stock of the breeder are sown and about 1000 plants conforming to varietal descriptors are delineated. Of these, about 200 plants are selected, from which seeds are harvested and threshed separately and examined for seed descriptors. Plant-to-row progenies from selected plants conforming to varietal descriptors are taken up retaining a portion of seed as reserve. The progeny rows are examined and those with confirmed characteristics are identified and reserve seed from the respective plants is bulked. Depending on the quantity needed for breeder seed production, one more season may be taken up and bulked seed forms nucleus seed.

3 Measures to Evaluate Varietal Purity to Increase Homogeneity and Stability

Maintaining trueness to type, so that genetic purity does not get affected during varietal maintenance and cycles of seed multiplication due to out-crossing, mechanical admixtures, residual segregation and mutations, has to be tackled by scientific means of planning like isolation, field inspection, checking for varietal descriptors during different stages. Examination of seeds, seedlings, control plot testing and varied biochemical and molecular mechanisms can be used for ascertaining the presence of delineated traits contributing to varietal purity.

Morphological characters (colour, pigmentation, appendages, etc.) of the seeds, seedling characters (coleoptile anthocyanin colour, plant growth habit, etc.), application of biochemical tests (phenol colour reaction, peroxidase test and electrophoresis techniques) and molecular markers can be deployed if necessary, to ensure and evaluate purity and to draw inferences in varietal maintenance programme. As the Indian DUS testing system is widely adapted from the UPOV list of characteristics, these are equally relevant to the Indian system of characterization of varieties.

Inferences on varietal purity can be drawn by resorting to some tests mentioned in Table 6 as per International Union for the Protection of New Varieties of Plants (UPOV 2017) and International Seed Testing Association (ISTA 2022) validated methods for species and variety testing. Some of these methods can be deployed even before sowing and can have an estimate of varietal purity. Advent of biochemical and molecular means of varietal testing widened the possibilities of application, as environment-dependent expression associated with some of the morphological parameters can be surpassed and can be used effectively in variety maintenance programmes.

Table 6 Examples of validated methods for species and variety purity testing (UPOV 2017, ISTA 2022)

Isolation is a powerful technique often practised by breeders during maintenance breeding, so that cross-contamination is eliminated. Isolation can be done via sowing time, local or spatial, or via artificial isolation, e.g. bagging, plastic walls or pollination nets. Insulation should be stringently followed, particularly in maintenance breeding programme of the A line, by use of separate equipment for each genotype, and wearing protective gear.

4 Conclusion

Variety development, maintenance and seed production represent a continuum. For the success of any variety development and seed production programme, maintenance breeding plays a critical role to get the most buck from every penny spent on crop breeding and seed production.