Certainly tobacco has played a major role in the economic development of Zimbabwe so far and, despite the health risks for tobacco consumers, it would be irresponsible to suggest that Zimbabwe should curtail tobacco and not continue to make use of this potential. More than 80% of all rose exports, for example, are grown on LSC tobacco farms and were first introduced using tobacco income. Similarly, 55% of the total area now under coffee is on tobacco farms including 5 500 hectares planted within the past three years. Zimbabwe’s LSC farmers are fully aware of the building pressure on tobacco markets and that favourable outlets for this crop are unlikely to last forever. Towards this end, tobacco growers have already taken steps to diversify their income base by investing in various new enterprises. This process should be supported with constructive domestic and international trade policies that help maintain tobacco and also encourage investment in new areas.In this respect, the results of the analysis are encouraging and show that several farm enterprises apart from tobacco offer a similar potential for good financial returns and high levels of employment creation. Coffee, roses, paprika and export vegetables can all be grown in the same areas as tobacco with good opportunities for high producer profits and payment in foreign exchange. Several other enterprises apart from those covered here have also been introduced on tobacco farms in recent years including citrus crops, wildlife production and small-scale tourism for photographic and hunting safaris. Other niche crops like marigold, spices, mushrooms, medicinal plants and flower seeds also offer potential on a limited scale.Importantly,vertical grow towers each of these alternative crops is generally more difficult to market than tobacco with only limited international demand.

This is especially true with respect to paprika and supermarket vegetables, which can be grown using much of the same equipment already available on most LSC farms, but do not offer the same potential for broad participation by all farmers as tobacco. Most of the so-called diversification crops also require a large initial investment in special infrastructure and processing facilities. This is especially true for roses, which can generate more income on a per hectare basis than any other enterprise, but are extremely expensive to produce and can easily cost more than ZWD 23 million to establish for a typical two hectare project. Tree crops like coffee are less expensive than roses, but take several years to mature and so demand a long-term perspective from potential investors. For these and other reasons, it is unlikely that any single enterprise could ever substitute for tobacco on its own and the challenge for each farmer must be to find the right blend of enterprises that works best for them. With LSC farmers, for example, it is not difficult to imagine a future in which various crops like horticulture, coffee, paprika and other similar enterprises could play a similar anchor role to tobacco in a mixed farm system. Assuming key production and marketing constraints can be overcome , these export crops can all be highly profitable and could easily substitute for some of the foreign income and employment currently accounted for by tobacco. Like tobacco, however, these commodities are all expensive to produce and it is also probable that traditional field crops including maize, cotton, groundnuts, soybeans and wheat will still have an important role to play in terms of providing supplemental income and steady cash flow needed to sustain the overall system. Traditional crops are perhaps less important for a farm system based entirely around horticulture since these crops can generate a steady cash flow in their own right, and it is again important to stress that the optimal enterprise mix can vary greatly from farm to farm depending on each system’s own individual requirements and farmer objectives.

For smallholder tobacco farmers, the analysis shows the opportunities for growth and diversification away from tobacco are more limited. Paprika, coffee and export vegetables produced on an out grower basis all offer considerable potential, but still depend on new infrastructure and support services for farmer training and input supply. Whereas LSC farmers have been able and willing to finance this type of investment on their own using tobacco revenue, smallholder farmers generally do not have this capacity and will require support from both public and private sector participants to succeed with these new crops. In this respect, a return of economic and political stability to Zimbabwe is especially important as a prerequisite for broad-based growth and diversification. In commenting on the diversification opportunities for smallholder farmers it must also be recognised that these growers are only marginally involved in the tobacco sector and account for less than 5% of total annual production. More specifically, of the estimated 1.21 million SSC, communal and resettlement farmers operating in Zimbabwe, only about 16 000 of these actually grow tobacco. Finding remunerative alternative to tobacco for these households is still an important challenge, but certainly not on the scale of having to work with all smallholder farmers in all locations. In a stable macroeconomic environment, for example, it is not unreasonable to expect that private investors could help develop some of the support services and infrastructure smallholder farmers need for success with other high value crops like paprika, supermarket vegetables and coffee. More than 1 700 smallholder farmers in tobacco areas are already organised around water points to produce export vegetables on an out grower basis for one private firm and Zimbabwe should look to encourage these and other similar developments. In terms of the potential negative effects on smallholder farmers from a loss of tobacco revenue, therefore, the greatest impact over time is likely to be felt through reduced remittance payments from workers on large-scale commercial farms.

The quantitative analysis clearly demonstrates that each of the major smallholder crops covered here including maize, cotton, groundnuts and even soybeans are much more profitable when farmers are able to afford the fertilisers and agro-chemicals needed to produce using medium and high input management than with low input use. To the extent that remittance payments facilitate improved crop management, therefore, the loss of this income could seriously impact smallholder agriculture throughout the country. Clearly, one of the main challenges for Zimbabwe is to develop reliable supply networks that ensure affordable crop inputs are available in all areas. Although many of the so-called diversification crops being introduced on LSC farms are also very labour intensive and so could provide a reliable income for some of the workers displaced by a gradual shift away from tobacco, Zimbabwe cannot afford to ignore the role this traditional cash crop in helping to support smallholder farmers.Many other conclusions besides those noted above can be drawn from the detailed information presented in the narrative text and quantitative data section. Agricultural administrators, farmers, policy makers,vertical growing racks agribusiness firms and others are all likely to interpret the data differently with an increased emphasis on their particular area of concern. Once a basic set of enterprise models have been prepared, however, it is very easy to use computer software to test the effects of alternative yield and price assumptions. At the very least, it is hoped this discussion has helped to illustrate the benefits of this approach to agriculture sector analysis. In the absence of a well defined methodology for assessing individual crop attributes, sector planning can easily become an exercise in guess work based on presuppositions about which crops and policy initiatives are best. The approach adopted here cannot point to optimal farm strategies, but can help to interpret some of the trade-offs and decisions farmers must make. The woody plant pomegranate produces colorful flowers and fruits with ornamental and culinary values. Different pomegranate tissues have historically been used for alleviating symptoms or treating various diseases due to the accumulation of a wide diversity of bio-active metabolites. In recent years, pomegranate fruits and juice have been pursued by consumers for their favorable nutritional quality, contributed by the abundant phenolic compounds, e.g., hydrolyzable tannins and flavonoids, in these tissues and products. Genetic variations underlying different metabolite profiles reportedly exist in pomegranate and have been utilized for breeding cultivars with desirable traits. Complementary to the classic breeding approach, new molecular techniques, such as genome editing, can enable targeted modification of key metabolic genes for improved nutritional and commercial quality of pomegranate fruits and products. Among the various genome-editing technologies, Clustered Regularly Interspaced Short Palindromic Repeats /CRISPR-associated protein 9 has gained increasing popularity for its efficiency and ease of use. In this method, a single guide RNA directs the Cas9 nuclease to the target gene sequence upstream of a protospacer adjacent motif. Cas9 creates a break in the double-strand DNA, which is then ligated by homology-directed repair or non-homologousend joining. In general, five genotypes can be obtained from the CRISPR/Cas9-mediated genome editing in a diploid species, including wild type , homozygous mutant , heterozygous mutant/monoallelic , biallelic , and chimera. Initially used for disruption of gene function, there has been rapid advancement in the CRISPR/Cas9 technology for more precise and versatile genome editing. Although CRISPR/Cas9 has been successfully adopted in many plant species , its application has not been reported in pomegranate. In consideration of the time and effort required for transformation and regeneration of pomegranate plants, we chose a hairy root system for testing the feasibility and efficacy of CRISPR/Cas9-mediated genome editing in pomegranate.

This is because hairy roots can be induced from different pomegranate explants, accumulate HTs and other phenolic compounds, are transformable, and produce sufficient amounts of tissues for molecular and metabolite analyses within 3 months of transformation. To select an easily discernable phenotype for verification of successful genome editing in pomegranate hairy roots, we chose PgUGT84A23 and PgUGT84A24, encoding two UDP-dependent glycosyltransferases that form β-glucogallin from gallic acid and UDP-glucose, as target genes. Reduced accumulation of punicalagin α and β isomers was observed in pomegranate hairy roots with attenuated PgUGT84A23 and PgUGT84A24 activities.Therefore, the punicalagin levels in hairy roots can serve as a metabolic phenotype for knocking out PgUGT84A23 and PgUGT84A24 activities through genome editing. In this work, we generated pomegranate hairy root lines containing CRISPR/Cas9-edited PgUGT84A23 and/or PgUGT84A24. We also modified the expression plasmids by incorporating a green fluorescent protein marker for rapid and non-destructive screening of transgenic hairy roots. Metabolite analysis was conducted on the control as well as the single and dual CRISPR/Cas9-edited hairy roots and showed significant changes in ugt84a23 ugt84a24. Comparative transcriptome analysis was subsequently carried out on the control andugt84a23 ugt84a24 hairy roots, which led to the identification of a new regioselective UGT toward gallic acid.This study demonstrates that CRISPR/Cas9-based genome editing, A. rhizogenes-mediated hairy root transformation and non-destructive screening of transgenic hairy roots, as well as transcriptome analysis collectively enable efficient and effective gene discovery in pomegranate. The unique accumulation of gallic acid glucosides in the ugt84a23 ugt84a24 hairy roots also suggests that the CRISPR/Cas9 method holds the potential for developing new pomegranate cultivars with modified phytochemical profiles. The presence of gallic acid 3-Oand 4-O-glucosides has only been reported in fruits of blackcurrant, gooseberry, jostaberry, raspberry, blackberry, blueberry, Arbutus unedo , and grape. Therefore, it is interesting that gallic acid glucosides can also be found in a non-reproductive plant tissue. The CRISPR/Cas9-sgRNAs generated mismatches, inframe , or out-of-frame deletions, as well as insertions of 1-bp, 2-bp, or 7-bp in PgUGT84A23 and PgUGT84A24 . The above-mentioned insertions and the out-of-frame deletions are expected to result in a frameshift and incorrect translation of the protein. Because the mutant lines containing in-frame deletions exhibited metabolic phenotypes similar to those with insertions and out-of-frame deletions, it suggests that the missing amino acids resulting from the in-frame deletions play important roles in enzyme activities. In addition to the homozygous, monoallelic, and biallelic mutants, chimeras of more than two mutated alleles were also identified in the CRISPR/Cas9-edited hairy roots . It could be because the hairy roots induced at the inoculation sites contain heterogeneouscells of different CRISPR/Cas9-edited or non-edited gene alleles. To establish homogeneous hairy root clones, a single hairy root tip would need to be recultured in phytohormone-free growth medium for multiple rounds and then tested for homogeneity. On the other hand, the metabolic phenotype of ugt84a23ugt84a24 hairy roots indicates the advantage of metabolite profiling in detecting knockout of enzyme activities in a heterogeneous cell population.