Authors: V. Poobalan*1, R. Muthuselvi1 and Deepa1
1Research Scholar, Horticultural College & Research Institute, Tamil Nadu Agricultural University, Coimbatore, India
*Corresponding author: poobalanagri007@gmail.com
Introduction
- Chilli pepper is an important horticultural crop that can surely benefit from plant biotechnology.
- However, although it is a Solanaceous member, developments in plant cell, tissue and organ culture, as well as on plant genetic transformation, have lagged far behind those achieved for other members of the same family.
- Aimed at genetic improvement against pests and diseases.
- Capsicum members have been shown to be recalcitrant to differentiation and plant regeneration under in vitro conditions, which in turn makes it very difficult or inefficient to apply recombinant DNA technologies via genetic transformation
- Biotechnology techniques involving plant tissue culture and recombinant DNA technologies are powerful tools that can complement conventional breeding and expedite Capsicum improvement.
Problems associated with Capsicum biotechnology
- Preliminary studies have vividly indicated various inherent problems associated with in vitro studies of Capsicum such as severe recalcitrant morphogenic nature.
- Formation of rosette shoots or ill-defined shoot buds, genotypic dependence,
- Which alone or together jeopardize the whole tissue culture efforts and in turn plant improvement through genetic transformation.
Severe recalcitrant morphogenic nature
- Recalcitrance is the inability of plant cells, tissues and organs to respond to in vitro culture. Tissue culture responses are greatly influenced by three main factors viz. whole plant physiology of donor, in vitro manipulation, and in vitro stress physiology (Benson, 2000).
Formation of rosette shoots or ill-defined shoot buds
- One of the most important limiting factors for regeneration of cultures in Capsicum is the formation of rosette shoots or ill-defined leafy shoots which do not elongate or resist elongation.
- Several inroads aimed to overcome difficulties of shoot elongation have been examined. Arroyo and Revilla (1991) directly rooted the rosette of buds and elongation took place in the field itself.
Plant regeneration systems
- In vitro culture is one of the key tools of plant biotechnology that exploits the totipotent nature of plant cells, a concept proposed by (1902) and unequivocally demonstrated for the first time by Steward et al. (1958).
1. Organogenesis
- Organogenesis is a complex phenomenon involving the de novo formation of organs (shoots or roots).
- Shoots can be derived either through differentiation of non-meristematic tissues known as adventitious shoot formation or through pre-existing meristematic tissues known as axillary shoot formation.
- Both these approaches require synergistic interaction of physical and chemical factors
a) Effect of genotype/cultivar
- Genotype is one of the main factors that influence the organogenic response of cultures in different plant species.
- Venkataiah et al. (2003) reported thidiazuron (TDZ)-mediated organogenesis in 10 pepper cultivars and the extent of the response depended upon the genotype specifically Of the 10 genotypes tested Capsicum annuum cv. CA 960, G4 and X-235 produced maximum number of adventitious shoots.
b) Choice of the explant
- Growth and morphogenesis of the plant tissue under in vitro conditions are largely governed by the appropriate choice of the explant.
- Analogous to this, pepper regeneration is also dependent on the age and type of the explant involved.
- Different explants including cotyledons, hypocotyls, leaves, shoot tips, zygotic embryos, embryonal leaves, stems, internodes, mature seeds and roots have been employed for plant regeneration in Capsicum
c)Effect of plant growth regulators and growth adjuvants on regeneration in Capsicum
- Shoot regeneration in Capsicum species is also highly influenced by the media formulations containing plant hormones and other growth regulators.
- Gunay and Rao (1978) for the first time tested different hormonal regimes for organogenesis in Capsicum.
d)Other factors including carbon source, light regime, gelling agent and temperature
- In vitro plant regeneration requires the source of carbon for flourishing and development of shoot buds. Sucrose 3% is commonly used as a source of carbohydrate.
- In general, for tissue culture Murashige and Skoog (1962) have stated that the use of 3% sucrose is better than 2% or 4%.
- However, there are many accounts on the use of other carbohydrate sources for shoot initiation and proliferation in Capsicum.
2. Somatic embryogenesis
- Somatic embryogenesis plays an important role in clonal propagation when integrated with a conventional breeding program and molecular and cell biology techniques.
- Somatic embryogenesis provides a valuable tool to enhance the pace of genetic improvement of commercial crop species (Stasolla and Yeung, 2003)
- A protocol for the separation of somatic embryos from embryogenic suspension cultures based on a cold treatment has been reported by Buyukalaca et al. (2003).
- Direct somatic embryogenesis and plant regeneration from stem segments and shoot tips of C. annuum on TDZ supplemented medium have been described by Khan et al. (2006).
3. Anther culture for androgenic haploid production
- The pioneering report of regeneration of haploid plants through anther culture was given by Guha and Maheshwari (1964) in Datura innoxia.
4. Protoplast-to-plant regeneration
- The importance of protoplast cultures resides in the possibility of recovering somaclonal variants, somatic hybrids in cases of sexual interspecific incompatibility and F1 hybrid sterility.
- There are only a few reports dealing with plant regeneration from protoplasts.
- Although chilli pepper protoplasts can be obtained easily, subsequent cell division, and plant regeneration have been found to be rare events.
- Factors influencing protoplast isolation and culture in C. annuum, C. baccatum and C. chacoense have been studied by Lim and Lian (2001).
- They were successful in isolating protoplast from cotyledon, hypocotyls and mesophyll tissues using a combination of cellulysin (1%), macerozyme (0.25%) and 0.65 M sorbitol.
- Antioxidant MES in the enzyme solution helped to overcome browning of protoplast
Genetic transformation
- There is also a current concern on the possibility of gene flow from genetically modified (GM) to conventional chilli peppers.
- PCR analysis (35S terminator)was also performed to confirmthe presence of transgenes in the kanamycinresistant seedlings. It was found that from a total of 11,194 ‘P915’ seeds, 67 were hybrids; and there were 40 hybrids of 7499 seeds, and 102 hybrids of 5340 seeds for ‘Manidda’ and ‘Taesdan’, respectively.
- In general, the gene flow frequency was low, but values as high as 17.89% between GM and ‘Taesan’ chilli pepper were observed at the closest distance (0.5 m) from the central GM plot.
In vitro plant regeneration in Capsicum chinense
- An in vitro plantlet regeneration protocol was developed for Capsicum chinense Jacq. Naga Chili, one of the world’s hottest chili cultivars and an important horticultural crop of Northeast India
- In vitro propagation plays a major role in conservation of genetically pure plants, crop improvement and production of disease free planting materials.
- effect of different compositions of plant growth regulators on multiple shoot development and callus induction was investigated.
- Multiple shoot was induced by culturing explants in MS medium supplemented with Benzyl adenine (BA) in combination with Indole-3-acetic acid (IAA).
- Maximum numbers of shoot buds were induced in MS medium containing 5 mgl-1 BA and 0.5 mgl-1 IAA. Successful induction of callus from stem segments of in vitro raised plants were achieved in MS medium in combination with 3 mgl-1Benzyl adenine (BA) and 1 mgl-1 1-Naphthaleneacetic acid (NAA).
- Shoot elongation and rooting were achieved in MS basal medium. This is the first successful report of plant regeneration from calluses in Capsicum chinense Jacq. Naga Chili.
- This protocol can be used as a cost effective method for the production of disease free planting materials and for genetic improvement and conservation of the crop.
SCREENING OF CHILLI GENOTYPES FOR DROUGHT TOLERANCE
- Drought stress is one of the major abiotic stresses constraining plant productivity with apparent effect on plant growth.
- Identification of drought tolerant varieties and incorporating in plant breeding programmes to enhance productivity is very much important.
- So, in order to study the effect of drought on Capsicum annuum, a commercially important crop, effect of drought stress on proline content, chlorophyll content, relative water content and root lengths of eleven Capsicum annuum varieties were investigated.
- Drought stress was imposed at both vegetative and reproductive phases by withholding water for 10 and 20 days.
- Drought stress resulted in the increase of proline content and root length while chlorophyll content and relative content were decreased in all the varieties
- Among eleven varieties, two varieties No5 and S10 showed significant increase in proline content and root length, thereby resulting in more relative water content when compared with other varieties.
- Furthermore results revealed that there was less damage to chlorophyll in these two varieties. (Lakshmi Sahitya Uppuluri1 and Krishna MSR)
Conclusions
- Significant progress aimed towards the genetic improvement of Capsicum has been made, but still there is a long way to go in this direction.
- Important advances have been materialized in pepper regeneration system and genetic transformation;
- The immergence and establishment of transformation techniques and perfection of the current procedures will be of enormous value as a tool for genetic improvement of Capsicum against various diseases caused by phytopathogenic fungi, bacteria and viruses to make a commercial impact on agriculture industry.
- Considerable efforts have been made to combat various types of diseases with special emphasis on virus-induced protection, but the results have not been quite satisfactory.
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