The following points highlight the two techniques adopted for genetic modified of tropical fruits. The techniques are: 1. Micro-Propagation 2. Embryo Culture Techniques.
1. Micro-Propagation Technique:
Micro grafting, an innovative technique which should be applicable to other tropical and subtropical species, was developed in 1972 for Citrus. Micro grafting of Citrus results in virus-free plants that are not juvenile. Most of this research is at a preliminary stage and this is demonstrated by two factors.
Firstly, with a few exceptions (citrus, banana, papaya, pineapple), most of the successes have been achieved using juvenile bud explants, and in many cases seedling tissue.
Secondly, only 50% of the reports document acclimatization of plantlets in soil.
However, the increase in research effort in this area in 1990’s and published results on a wide range of tropical fruit species is encouraging and justifies a larger research effort. Regeneration of plantlets from callus can follow two distinct pathways.
Organogenesis involves the development of meristematic tissue which produces either shoots or roots, with vascular connections to the parental callus. Somatic embryos progress through characteristic developmental stages (globular, heart, torpedo and mature), have both a shoot and root meristem (bipolar) and become independent from the parental callus.
As with most species, tropical and subtropical fruit species tend to regenerate by either organogenesis or embryogenesis, although occasionally there are reports of regeneration via both pathways (e.g.. Carica papaya). As with micro propagation, most of the published successes via regeneration from callus has been achieved using juvenile tissue explants.
Most tropical fruit species are woody perennial dicots, which in general are a difficult group to culture in vitro. Tissue culture of recalcitrant species has been achieved in many cases using immature embryos or very juvenile seedling explants, as these often represent the most regenerative tissue in a plant.
Much of the documented research on recalcitrant species entails a thorough evaluation of a range of media components and concentrations. However, recent innovative protocols for embryogenesis and regeneration of Musa spp. from male flower explants demonstrate the importance of testing a range of explants as well as media when working with untried or ‘difficult’ species.
However, few species are truly recalcitrant, and most can be regenerated provided they can be subjected to a concentrated research effort. In vitro culture of papaya has been researched extensively worldwide and highly refined protocols have been developed. Efficient methods for embryogenesis from immature zygotic embryos have been used to develop transgenic plants and encapsulated artificial seeds.
Recent notes of culture of integuments of immature seeds and the development of embryos of maternal origin should have important implications for rapid propagation and production of transformed ‘elite’ papaya genotypes.
Regeneration of plantlets via embryogenesis following culture of nuclear tissue of mango, another recalcitrant species, demonstrates what can be achieved by innovative research. There are two other noteworthy achievements with regeneration of tropical fruit species.
Plantlets have been produced after embryogenesis from mature leaf tissue of mangosteen, and seedless triploid pummelo plants were produced following culture of endosperm.
Regeneration via organogenesis or embryogenesis is seen as having three major applications: micro propagation, cryopreservation and gene transfer. However, regeneration from callus can be prone to production of genetic off-types, thus any micro propagation technique should not be assumed to be clonal until large scale field testing has been undertaken to evaluate a protocol for genetic stability.
Similarly, embryo genic cultures are well suited to cryopreservation studies, however an assessment of genetic stability after large scale field evaluation is also necessary for each species and protocol, before it can be endorsed as a method of germplasm conservation.
Regeneration protocols are basic to the development of transformation systems, as transformed plantlets have to be grown from single transformed cells. Thus a large research effort is warranted for many tropical and subtropical fruits, if.
2. Embryo Culture Techniques:
Production of plants from mature embryos is one of the most straight forward in vitro techniques. Immature embryos represent the most re-generable tissue for many species. Consequently, culture of immature embryos has facilitated both in vitro regeneration of recalcitrant species and rescue of interspecific hybrids.
The potential of embryo culture for the regeneration of recalcitrant fruit species has been demonstrated with Litchi chinensis.
Lychee is one of the most difficult tropical fruit species to establish and grow in vitro. Kantharajah cultured immature embryos of lychee as small as 3mm in length. Ability to produce adventitious buds varied with genotype, however cultivar ‘Bengal’ produced 15 shoots after pre-treatment of immature embryos with BAP. Root formation was achieved with 65% of adventitious shoots, and resulting plantlets acclimatized.
Although protoplasts can be isolated from a range of tissues of almost any plant species, regeneration of plants from protoplasts is one of the most difficult in vitro techniques. Reports of success with recalcitrant woody species are limited, and applications to tropical and subtropical fruit species are rare.
The only exception is with Citrus species. There have been notes of protoplast isolation of non-woody tropical fruit species: papaw and banana, although sustained cell division was not achieved.
The principal uses of protoplasts are as targets for direct methods of gene transfer and to facilitate interspecific hybridisation. Direct methods of gene transfer requiring protoplasts are becoming obsolete. Although protoplast fusion may provide a method of interspecific hybridisation, the difficulties in achieving sustained cell division and plantlet regeneration in many species generally outweigh the advantages.
In reality, protoplast fusion is only an advantage when prezygotic barriers prevent interspecific hybridisation. If zygotic embryos are produced, embryo rescue and culture is a superior technique. A comparison of these techniques has been demonstrated with interspecific hybridisation of Carica papaya and related species.
An efficient method has been developed for embryo rescue, embryogenesis and plantlet production for hybridisation between C. papaya and C. cauliflora and between C. papaya and C. pubescens, C. quercifolia, C. parviflora and C. goudotiana.
By contrast, attempts to hybridise G papaya and C. pubescens have resulted in callus growth but no plants. Similar difficulties have been experienced in the authors laboratory when protoplasts of C. papaya were fused with those of C. pubescens, C. quercifolia and C. stipulata.
As with most difficult aspects of in vitro culture, intensive funding and research effort usually result in success. Protoplast culture of tropical and subtropical fruit species is no exception and, given sufficient attention, new and improved protocols would probably be developed. However, at this stage, it would be preferable to direct funds towards other aspects of in vitro culture that appear to hold greater potential.