**4. Improvement of tropical plants using innovative approaches**

#### **4.1 Cell and tissue culture**

Tropical plant communities contribute an enormous proportion of the global plant species and represent more than 42% of the total carbon reserves throughout the world [24]. They play a critical role in the well-being of humanity. Our daily dependence on tropical plants and/or their products is outstanding. They are not only major contributors to food and feed but also a valuable source of spices, essential oils, fruits, sugar, and beautiful hardwoods. In addition, tropical regions also produce different types of fibers, resins, gums, plant essences, and dyes, which are extensively used in therapeutics and various industrial by-products. They are the dominant source of trade among the continents; for example, Africa and Latin America are the major suppliers of cacao and coffee, South America is the largest producer of sugar and bioethanol, whereas Asia produces most of the natural rubber.

With the increased demand for tropical plants, that is, rubber, oil palm, cocoa, banana, pepper, and pineapple, their large-scale production is direly needed. Highquality planting material can only be produced through tissue culture, thus the only potential strategy to fulfill the increased demand for ornamental tropical plants and plants of economic value. So, different types of explants, cultural media, and conditions have been worked out by different research groups. Micropropagation by somatic embryogenesis has been established through direct or indirect routes. The number of clonal plants is usually lower in the case of direct embryogenesis as compared with indirect somatic embryogenesis, so indirect embryogenesis is preferred for the commercial-scale production of ornamental plants, particularly in the case of endangered plant species, where only limited explant material is available. However, callus induction and somatic embryogenesis are dependent on exogenous auxin and culture conditions [25]. Exogenous auxin is involved in the downregulation of essential genes involved in embryogenesis perhaps by DNA methylation or other cellular processes, which is still to be explored. Removal of auxin from the culture media supports embryogenesis in most plant species [26]. Exploring the developmental trajectory of callus induction, indirect somatic embryogenesis, and direct somatic embryogenesis will be of great help for the commercial-scale production of indoor tropical plants and edible tropical plant species. Different types of explants including

immature zygotic embryos, mature zygotic embryos, immature female inflorescence, immature male inflorescence, immature leaves, mature leaves, young plantlets, and shoots were tested for the micropropagation of oil palm *via* indirect somatic embryogenesis [27].

Advancements in molecular biology have not only explored the critical pathways and genes involved in different phases of *in vitro* growth but also led to manipulation of certain genes to improve somatic embryogenesis and regeneration. Though most of the research has been reported on *Arabidopsis*, yet these findings may be extended for the betterment of other plant species including tropical plants. LEAFY COTYLEDON genes (LEC1 and LEC2) were found to be involved in the key pathways of somatic embryogenesis, and their overexpression triggered the upregulation of YUC genes, which are responsible for the increase of endogenous auxin levels. Other valuable genes involved in somatic embryogenesis are BBM, WOX, and SERK. Genetic manipulation of these genes can be of great help to further explore and understand fundamental cellular processes involved in somatic embryogenesis and clonal propagation [28]. In addition, proteomics studies have helped to elucidate the fundamental processes involved in these crucial cell growth phases, thus helping out to promote plant growth under *in vitro* conditions. Three valuable proteins linked with somatic embryogenesis were identified as osmotin-like proteins, chitinase, and β-1,3 glucanase. In *Picea glauca*, 48 differentially expressed proteins were observed to be of crucial importance during different stages of the development of somatic embryos. Efforts have been made to explore the proteome profile of oil palm embryogenic lines, embryogenic cell suspensions of coffee [29], auxin-induced embryogenic and non-embryogenic tissues of tamarillo, secondary somatic embryogenesis in cassava, and somatic embryos of avocado, which could further be exploited to improve clonal propagation of these valuable tropical plant species [30].

Though efforts have been made to unravel the fundamental cellular processes involved in key regulatory pathways of cell differentiation, dedifferentiation and commercial scale production of tissue culture plants have been possible. Still, certain impediments are there that need to be addressed for further improvement of existing clonal propagation systems. These include the genotype-dependent nature of the cultures, slow response of the cultured tissues, lower conversion rate of embryonically competent tissues, and heterogeneity of the cultured samples.

#### **4.2 Genomics approaches**

Breeding of the tropical tree plants is complicated owing to polyembryony, parthenocarpy, long juvenile phase, polyploidy, generation cycle, heterozygosity, and insufficient genomic resources. Advancements in multi-omics approaches, that is, genomics, metabolomics, transcriptomics, proteomics, whole genome sequencing, and annotation, have led to the identification of novel genes involved in crucial metabolic pathways responsible for sugar metabolism, fruit development, fruit ripening, stress tolerance, shelf life, etc. Interventions in genome-wide association (GWAS), genomic selection (GS), genetic transformation, and genome editing through CRISPR/Cas9 have helped to develop tropical plant varieties with desirable traits.

Developments in sequencing techniques have not only helped researchers devise molecular markers but also paved the way to explore genetic diversity in different plant species. The world's largest germplasm collection of bananas has been maintained at Biodiversity International Transit Centre (ITC), Belgium [31]. Diversity arrays technology (DArT) was employed for the selection of carotenoid-rich

#### *Emerging Trends to Improve Tropical Plants: Biotechnological Interventions DOI: http://dx.doi.org/10.5772/intechopen.108532*

bananas, thus helping out to promote nutrient-enriched bananas [32]. In papaya, 21,231 SSR markers were developed from genic regions, of which 73 SSR markers were validated for fruit ripening. The SCAR marker (CPFC1) was developed for the fruit flesh color in papaya, facilitating the identification of progenies based on pulp color. Likewise, AFLP markers were developed for the characterization of pinkfleshed and white-fleshed guava genotypes. Comparative mapping and germplasm characterization were performed by SSR markers in Musa species. These studies helped out to shortlist the Musa accessions with relatively higher content of minerals including calcium (111.1-fold higher), potassium, and magnesium (4.7-fold higher) [33]. Jackfruit draft genome assembly has helped to explore numerous gene families involved in starch synthesis and fruit development. SSR and ISSR markers have also successfully been employed for the identification of dragon fruits with white pulp and pink pulp [34].

Molecular breeding, that is, marker-assisted selection (MAS), marker-assisted backcrossing (MAB), and marker-assisted introgression (MAI), is quite helpful in the efficient development of new genotypes, mapping population, phenotyping, and genotyping. QTLs (quantitative trait loci) are of pivotal importance in this context, to track particular desired traits. In papaya, 21 QTLs were identified for the key quality traits of fruits, that is, fruit weight, fruit width, fruit length, flesh thickness, flesh sweetness, fruit firmness, and skin freckle. Similarly, 460 SNPs were predicted as potential molecular markers for the selection of particular fruit traits and diversity studies [35]. In mandarin, four QTLs were identified to be associated with fruit weight, three with peel puffing, and one with sugar content. The whole genome assembly of mango has helped to explore polyembryony, identification of non-coding RNAs, and QTLs for flavonoid biosynthesis and fruit weight [36].

The discovery of genome-wide SNPs has opened up new avenues in high-throughput genotyping and marker-assisted breeding, thus helping out to develop the novel genotypes. These SNPs have been used for the GWAS and identification of QTLs relevant to fruit traits in tropical plants, that is, mango, papaya, avocado, cassava, banana. These QTLs can effectively be used for the betterment of traditional breeding in terms of reduced time and cost. In addition, information retrieved from the pan-genome, super-pan-genome, and pan-transcriptome has been employed in the mining of genetic determinants of various phenotypes, thus helping out the betterment of tropical plant species.

#### **4.3 Genetic engineering**

Transgenic technology is increasingly contributing to the betterment of tropical and sub-tropical plants. Numerous fruit trees, crop plants, and ornamental plant species have been engineered for valuable traits, esthetic value, and the cleanup of the environment. Compared with the annual crops, tree plants are tough targets as far as their genetic manipulation is concerned. The complexity of the genome, low transformation efficiency, complex cultivation environment, long breeding cycle, and recalcitrant nature of the plant tissues are the major impediments in the genetic transformation of tree plants. Researchers have worked out to resolve these bottlenecks, and protocols have been established for the genetic transformation of numerous tropical and subtropical plants including citrus, mango, banana, pineapple, litchi, passion fruit, plantain, longan, and avocado. These plant species have been engineered not only for valuable agronomic traits but also for the improved quality and quantity of the fruits.

*Populus alba* is taken as a model plant for the establishment of genetic transformation in tree plants. It has been engineered for insect resistance, herbicide tolerance, and decreased lignin content. Insect-resistant transgenic poplar plant has been approved for commercial-scale cultivation in China, wherein stable expression of the transgene was observed in 8- to 10-year-old transgenic plants, hence providing broad-spectrum resistance against insect pests [37]. Stable transformation of Cavendish banana cv. Grand Nain was also reported using *uidA* and the potential virus-resistance gene (BBTV) along with the *nptII* gene as a selectable marker expressed.

Diverse tropical plants including papaya, oil palm, cassava, Picea, Ulmus, and Pinus have been engineered for disease resistance, herbicide tolerance, and resistance against insect pests [38]. The first draft of the papaya genome sequence from the commercial virus-resistant transgenic fruit tree opened up new avenues for the genetic transformation of tree plants [39]. Papaya has a relatively small genome of 372 Mb, with nine pairs of chromosomes and diploid inheritance. Other desirable features of the papaya are short generation time (9–15 months), primitive sex chromosome system, and continuous flowering throughout the year. These features make papaya a promising system for the exploration of fruit tree genomics and tropical tree genomes.

The first commercialized transgenic papaya carrying coat protein for papaya ring spot virus, PRSV CP gene, was introduced in Hawaii in 1998. CP-transgenic papaya plants appeared to have variable levels of resistance against ring spot viral isolates from different geographical regions. Isolates from Florida, Bahamas, and Mexico have delayed and made symptoms mild, whereas isolates from Thailand and Brazil have delayed symptoms; as a result, the virus can overcome resistance and thus may cause pathogenicity. Rainbow, a hemizygous line, also appeared to be susceptible to viral isolates from Taiwan [40]. Hence, the level of resistance appeared to be different against isolates from different regions. Broad-spectrum resistance has also been attempted to develop through RNAi by targeting the conserved domain of the PRSV CP gene [41]. The disease-resistant transgenic papaya was reported to be environmentally safe, with no harmful effects on human health. Oil palm is another valuable tropical plant that has extensively been used for the production of edible oil. Parveez and Christou [42] reported its genetic transformation through a biolistic transformation using multiple gene(s) constructs, that is, gusA, bar, hpt under ubiquitin, and CaMV35S promoters. The resultant transformants were selected on 50 mg/L Basta. Bahariah et al. [43] published biolistic transformation, and resultant transformants were selected through a mannose selection system containing mannose @ 30 g/L. Oil palm genome has also been targeted for the production of polyhydroxybutyrate (biodegradable plastics). Three genes (phaB, bktB, and phaC) responsible for the bacterial PHB biosynthesis were expressed under the Ubi promoter. The expression of biodegradable plastic was detected to be in the range of 0.33 to 0.58 mg/g of dry weight. The transgenic oil palm plants showed normal growth; thus, no deleterious effects of transgene expression were observed on plant growth.

Cassava (*Manihot esculenta*) is the third major contributor of staple food in sub-Saharan Africa, where it is grown as a starch-storing root crop. It has been engineered for valuable agronomic traits as well as to boost its nutritional value. Zeoline protein has been expressed in roots wherein the total soluble protein was detected to be increased up to 12.5% of the dry weight, thus showing a fourfold increase in protein content as compared with non-transgenic plants. The Nigerian cultivars, TMS 91/02324 and TMS 95/0505, were engineered for resistance against CBSD and CMD.

#### *Emerging Trends to Improve Tropical Plants: Biotechnological Interventions DOI: http://dx.doi.org/10.5772/intechopen.108532*

The transformed cultivars showed an increased level of resistance against the noxious viral pathogens [44]. AtFER1 and AtIRT1were overexpressed in cassava for the increased accumulation of zinc and iron in roots (40 μg/g and 145 μg/g dry weight, respectively).

So, tropical plants have not only been engineered for improved agronomic performance and additive nutrients but also been engineered to clean up the indoor air and increase esthetic values. Indoor air often contains benzene, formaldehyde, chloroform, and other volatile organic compounds. Modern lifestyle has promoted the production of undesired molecules coming from furniture, smoking, and showering. A normal room needs at least 20 plants for the removal of these toxic molecules. The detoxifying ability of the plants can be increased through transgenic technology. Incorporation of mammalian cytochrome P450 2e1 (rabbit CYP2E1) in pothos ivy boosted its ability to detoxify the abovementioned hazardous molecules. The engineered plants were able to detoxify chloroform and benzene in the closed vials within 8 days of culture, thus showing great potential to detoxify the undesired molecules [45]. Likewise, sulfur metabolism can be engineered to improve resistance to SO2. Transgenic tobacco plants overexpressing serine acetyltransferase and cysteine synthase gene(s) were highly tolerant to sulfite and SO2 [46]. Engineering tropical plants with the said gene can uplift their ability to tolerate sulfite and sulfur dioxide.

#### **4.4 Role of biotechnology to secure endangered plant species**

Tropical plants occupy approximately 1/20th of the earth's surface [47]. They comprise 2/3rd of the terrestrial plant species globally. Most tropical regions are categorized into biodiversity hotspots, but they possess different challenges due to the increasing rate of the human population. The tropical biodiversity hotspots have increased habitat loss, species richness, and an increasing number of endemic species [48]. An important cause of these changes is deforestation due to agricultural and industrial expansion during the past 30 years. As a result, the diversity in the tropical ecosystem is endangered, which has high environmental concerns regarding biodiversity degradation and extinction of species. One example of a biodiversity hotspot is Sumatra, where ~0.84 million hectares of forest land has declined [49]. The same is happening in other developing countries. Intensified agriculture and changes in the use of tropical land have a durable impact on the global biodiversity, and their future consequences are just estimated.

It has been studied by Rodriguez-Echeverry et al. [50] that fragmentation, changes in land use, and habitat loss are causing the decline of biodiversity and the composition of species is changing. The ecosystem processes and invasive species are altering. In the past decade, various studies conducted to find its impact on the genetic composition of tropical plant species and genetic alterations were observed [51]. Habitat loss, population differentiation, and genetic diversity losses have consequences that are caused by inbreeding, genetic drift, and increased distances by isolation [52]. The outcome of these changes is also caused by different life traits such as dispersal strategy, the density of plant species, mating, and gene flow in tropical lands. The information on genetic resources collected from one of the few species cannot reflect the plant community. However, genetics and molecular biology provide efficient approaches to precisely calculate the genetic variability in tropical plants.

Conservation management is an important issue in the tropical ecosystem. Various human and social factors have been identified that are responsible for biodiversity loss [51]. Agriculture expansion, corruption, human growth, and incompetency in the

development of genetic conservation strategies have increased the risk to the tropical ecosystem and sustainable management. The genetic information of the tropical species has increased the probability to maintain the genetic conservation of targeted plant species. The recommendations on the monitoring program and sustainable management in a particular fragment of tropical land are based on the genetic information of species, species richness, and processes of the ecosystem. Lack of sufficient genetic study along with population fragmentation data of different plant species could lead to the development of poor management practices for the conservation of plant species. The land-use change process is fast in the tropics, and there is a need for a robust method for identification of biodiversity hotspots and development of strategy in the identified area. Genetics alone does not provide a sufficient solution for the determination of the hotspots in the tropical plant community. Therefore, the emerging molecular and biotechnological techniques provide the required solutions to identify the genetic diversity in the high number of plants. These biotechnologybased tools provide the pre-requisite baseline information of the dominant species composition. It also helps to identify the high conservation value of different habitats that could be used in the conservative management practices of endangered species.

One important technique to investigate multiple plant species is amplified fragment length polymorphism (AFLP). After DNA extraction of the selected plant species from nearly 1 cm<sup>2</sup> leaf tissues of each plant, the AFLP protocol of Vos et al. [53] provides better results. The extra genetic material can be stored at −20°C for a longer period at optimized conditions. The samples were excised with the single enzyme to incubate overnight or to amplify with a single primer. In this regard, different PCR protocols are optimized for different plant species [54]. For the efficient reproducibility of the AFLP procedure, two to ten samples of each plant species provide reliable information. The fragments occurring from the restriction steps in the repetition of samples are considered important. The results obtained from the AFLP linked with PCR techniques can be visualized by transforming into the fragment presenceabsence matrix. The analysis can be done by collecting samples of more than one hundred genotyped species. Different values of variation per species use the common genetic diversity indices [55].

*In vitro* conservation of germplasm included a large number of techniques involving the incubation of plant germplasm under controlled conditions. Although the nutrient requirement and conditions of growth vary for each tropical plant species, it provides a robust and reliable solution for the conservation of endangered species in the tropical region [56]. More commonly, the younger developing tissues of the explant can be used as the source. Genetic transformation has become an important tool in the additive one-point improvement in comparison with the mutation breeding that develops the subtractive one-point improvement. The genetically modified ornamental plants can be more acceptable to the consumers due to their vibrant colors as compared to food crops, where different ethical concerns are present regarding the use of recombinant technology [57]. Different GM technologies include zinc finger proteins, RNA interference, miRNAs, and CRISPR-Cas-9, which can be applied in the development of transgenic plants (**Figure 1**). The quality of the explant and type of tissue help to develop the biotechnology-based strategy for the regeneration of endangered species. The genome-editing technology has been vastly improved in the previous years. After the advent of genome-wide scan analysis and next-generation sequencing, varied information becomes available on tropical indoor and outdoor plants. The available information on the sequences of tropical plants could provide great help in breeding and basic research.

*Emerging Trends to Improve Tropical Plants: Biotechnological Interventions DOI: http://dx.doi.org/10.5772/intechopen.108532*

**Figure 1.**

*Contribution of biotechnology to the improvement of germplasm and agriculture in tropical areas. The DNA of tropical plants can be genetically engineered for the improvement of different traits, conservation of endangered species, and resistance against pathogens.*

The heterogeneity of the different biodiversity plots and land-use systems can be studied by using the fragment distance matrix that is based on the principal component analysis. The bioinformatics and statistical tools can be used by applying the function betadisper and R-package ggplot2. Precise estimation of the genetic diversity in an area of dominant species depends on the spatial scales of alpha, beta, and gamma. The alpha scales respond to the diversity within the plot, the beta scale is for the land-use system, while the gamma scale is the highest level of spatial scales. In the fragment pool approach, the differentiation at the alpha level is determined by using at least 10 fragment pairs within each plot. However, the β diversity level can be calculated by taking at least 40 pairwise fragments in every land system. On the other side, the γ diversity is based on using the 160 genetic fragments from the plots where the concerned species are dominant. To apply this technique, the Shannon Index can be applied within each plot.
