In this article we will discuss about the genetic engineering strategy for the production of male sterility plants.
Extensive progress has been made in cell and tissue specific gene expressing and discovery of novel genes which can potentially inflict tissue ablation through genetic engineering led to the successful production of male sterile plants partially or completely. The basic concept adapted in the strategy is to target the expression of gene encoding a cytotoxic driven under the control of an anther specific promoter.
Tapetal cells are targeted and make them dysfunction or breakdown of the key molecules inside the tapetal cells leading to non-functional pollen grain, which result in male sterile lines.
According to the studies following reports have been implicated in the production of male-sterile plants:
(a) Tapetum specific expression of cytotoxic degradation enzyme using barnase RNase system.
(b) Premature dissolution of microsporocyte callose wall leading to male sterility.
(c) Inhibition of particular enzyme or protein production by antisense technology.
(d) Induction of mitochondrial dysfunction by transgenic expression of unedited RNA.
In one of the earliest transgenic works on induction of male sterility, synthetic gene RNA-T1 from the fungus Aspergillus oryzae have been employed by Quacus (1998). In another attempt premature dissolution of the callose wall result in male sterility was observed in transgenic tobacco.
Before meiosis, (in angiosperms) microspores synthesise callose deposition continues through the meiosis and microspore is surrounded by callose. The callose wall is breakdown after meiotic process. For example, male sterility in petunia has been attributed to the early appearance of active callose and β-1.3-glucanase in the anther locule.
As a consequence, this led to premature dissolution of callose wall surrounding the microspore cells. Transgenic tobacco expressing modified glucanase from tapetum specific promoters led to premature dissolution of callose after prophase I of meiotic stage. Transgenic tobacco exhibit reduced male sterility, ranging from complete to partial male sterility.
This demonstrates that premature callose degradation is sufficient to cause male sterility and suggest that callose is indispensable for the formation of normal microspore cell wall. The callose is not exactly part of the cell wall but it participates in the formation of physical barrier against pathogenic attack and also prevents all cohesion and fusion.
TA 29 Barnase Male Sterility:
One of the most effective and classic case of male sterility induction was carried out by the complete destruction of RNA molecule in tapetal cells. It was demonstrated that chimaeric RNase and barnase gene driven under the control of tapetum specific promoter TA 29.
Where it can induce male sterility in tobacco and oil seed rape. The tissue specific TA 29 gene is highly regulated and specifically expressed in tapetum cells of anther. Expression of TA 29 barnase fused gene consequently acts as cytotoxic and selectively destroys the tapetal cell layer by complete elimination of RNA molecule.
Tapetal cell is a nutritive layer which surrounds the pollen sac in the anther pollen development requiring nutrition obtained from tapetal cells. Therefore, destruction of tapetal cells cut off food supply, arrest pollen development or non-functional pollen will be formed to produce sterile plants.
The destruction of tapetum by RNase and RNase T1 gene derived from bacterium Bacillus amyloliquefaciens and fungus Aspergillus oryzae, respectively. The same TA 29 RNase gene combination induced sterility was also produced in maize and other vegetable species.
TA 29 Barstar Male Fertility:
Expression of a novel gene known as barstar derived from the same bacterium Bacillus amyloliquefaciens lead to the production of normal male fertile plant without interfering its pollen development. Bacillus amyloliquefaciens expressing barnase gene has corresponding inhibitor protein called barstar. Barstar is produced intracellularly and protect bacteria from the harmful role played by barnase by forming stable complex with the barnase in the cytoplasm.
The gene 1.5 kilobase barstar was fused to tapetum specific TA 29 gene upstream fragment that comprises all regulatory segments targeted for tapetum specific expression. Mariani et al., 1992 introduced TA 29 barstar into oil seed rape by employing Agrobacterium Ti plasmid and biolophase selectable marker gene. Barstar expressing transformants were male fertile, produced normal anther with well-developed anther tapetal cells.
Fertility Restoration by Barnase-Barstar System:
Once male sterility is induced, plants are then can be exploited for introduction of hybrid seed production and plants are maintained as male sterile lines. Restoration of fertility has been accomplished by crossing male sterile plants with male fertile plants expressing barstar gene.
To determine whether TA 29 barstar gene expression could inhibit barnase activity in tapetal cells and led to male fertile restoration, Mariami selected TA 29 barstar gene containing male fertile plants and crosses with male sterile parental plants expressing TA 29 baranse single copy germlines.
Both barnase and barstar gene co-expressed specifically in the tapetal cells of anther led to the formation of barstar-barnase stable complex. In the Fi progeny ratio of fertile to sterile would be 2:1 ratio. All F1 progeny from the crosses that were expressing both chimeric genes were male fertile led to the conclusion that TA 29 barstar gene functioned as a dominant suppressor of TA 29 barnase gene activity (Fig. 21.2).
Plants which are restored to fertility are indistinguish from those of wild-type plants develop normally and exhibited normal anther dehiscent process. They have well developed tapetal cell layers and produce vast amount of functional pollen grain. Thus, effectiveness of chimeric TA 29 barnase and TA 29 barstar genes system in the male-sterility induction and male fertility restoration may permit the breeding of genetic engineering hybrid plant.
Transgenic lines exhibiting the barstar gene have also been developed in Indian Mustard, Brassica juncea, to develop a complete male sterility and restoration system for heterosis in this crop. Transgenic mustard lines containing a modified barstar were also raised using modified sequence of the barstar gene.
Another example of obtaining parental male sterility in transgenic tobacco by blocking the synthesis of flavanols and starch in pollen grain has been reported by Mutsuda (1996). Reduction in levels of flavanols and sporopollenin of the pollen wall has been demonstrated to be associated with abnormal microspores and abnormal growth of pollen tube led to reduced fertility.
Pollen cells have plenty of actin which is an essential protein in cell function, blocking the production of actin by antisense actin gene expression leading to male sterile plants in crop plants.
Phenyl ammonia lyase, key enzyme (PAL) in phenyl propanoid pathway, responsible for the synthesis of flavanoids. Introduction of sense or antisense PAL cDNA of sweet potato under the control of tapetum specific rice promoter into tobacco generated transgenic plants. This exhibited reduced pollen fertility. The pollen fertility of these plants droped from 8% to 6%.
Reduction of PAL activity in anther was correlated with the number of fertile pollen grain at flowering stage. These results demonstrated that manipulation of phenyl propanoid pathway by transgene provides a powerful tool for a alternative of phenyl propanoid in pollen and lead to reduced fertility.
Molecular control of male fertility has been studied in Brassica. The gene, BcP1, essential for production of functional pollen grain has been manipulated by their expression using antisense approach. The specific down regulation of BcP1 further demonstrated the importance of this gene during pollen development.
Transgenic Arabidopsis plants in which the BcP1 gene expression is blocked either in tapetum or in developing microspore using Lat52 promoter show arrest in pollen development leading to pollen abortion and exhibit male sterile phenotype.
Apart from antisense approach or production of degradative enzymes for flavanoid biosynthesis, deploying unedited gene targeted to tapetal mitochondria induces male sterility. The expression of transgene, designed to contain the unedited atp 9 mitochondrial gene (u atp 9) fused to the yeast COXTV led to the production of male sterile plants. In contrast expression of edited transgene in the same plant led to the production of male fertile plants with normal pollen development.
RNA editing in plant mitochondria is a post-transcription process improves protein synthesis of mitochondrial proteins which are basically derived from edited mRNA. However, several exceptions have been recorded. Expression of unedited gene led to the production of non-functional proteins and produce male sterile plants.
The modification of mitochondria was correlated with presence of translated product of unedited atp 9 and a significant decrease in oxygen consumption in non-photosynthetic tissue. The high reduction of CO2 consumption in root meristems of male sterile plants confirms improvement of mitochodrial function.
Due to the drop in respiration, transgenic male sterile plants are unable to maintain the high energy level (ATP) required for anther development and micro-sporogenesis. These factors suggested that RNA editing is indispensable in the production of functional proteins.
Carbohydrate was shown to play a critical role in anther and pollen development. According to studies, different male sterile lines were shown to be characterised by altered carbohydrate metabolism. Once carbohydrates are produced in source region, they are transported to sink region or inactive sink tissues by unloading pathway.
An extracellular invertase bound to cell wall hydrolyses to transport sucrose. The importance of extracellular invertase to assimilate partitioning and source sink regulation by supplying carbohydrate to sink tissues via the apoplast has been suggested. The specific interference with phloem unloading at metabolic signalling during pollen formation will be a potential valuable approach to induce male sterility in various crop plants.
Goet (2001) reported cloning of a gene encoding an extracellular invertase isoenzyme from tobacco that shows a specific spatial and temporal expression in anthers. Transgenic tobacco plants transformed with an antisense construct of extracellular invertase under its own promoter are blocked in early pollen development, thus, causing male sterility. Several male sterile genes expressed in transgenic plants for the production of male sterile plants has been summarised in the Table 21.1.
