A Review on In-vitro Haploid Production in Orchids

The aim of writing this paper is to review production of haploids in orchids in vitro. Haploids possess half number of chromosomes and do not undergo fertilization. In vitro conditions provide necessary nutrients and conditions that are required for growth of haploid plantlets. The natural breeding cycle of orchids is very slow as well as unpredictable. To reduce this time, the technique of in vitro haploid production is used. In vitro conditions can decrease or shorten the time required for juvenile period in Orchids. Haploid plants are produced from in vitro haploid culture. This technique is useful to produce homozygous pure lines and to increase the yield of a plant.


INTRODUCTION
Orchids are the most favourite as well as the most expensive cut-flowers. They are commercially very valuable. Due to the high market value of Orchids, there would be pressure on the cultivators to produce a large number of Orchids. The only hurdle in this process is the natural breeding program of Orchids. Family Orchidaceae contains very beautiful and vibrant flowers. Orchids are found almost all over the world, except in the freezing polar regions and extremely hot deserts. Orchids are mostly found in tropical forests [1]. The flowers of the Orchidaceae family are of different shapes and radiant colours [2]. Orchidaceae is a very interesting family because of the colours of its flowers and the exceptional structures of the flowers [1]. These flowers are commercially very useful. Orchids flowers have high artistic value.

Review Article
Orchids are endowed with economic worth at par with other ornamentals [3].
Orchidaceae contains roughly 35,000 species, in 1,000 genera and 1,00,000 hybrids [4]. Orchids are around 8% to 10% of all flowering plants [4]. The annual orchid flowers production in India is approximately 1000 tonnes making a small share of 0.01% in international market [2,5]. Orchids are the most expensive cut-flowers. Besides their ornamental value, some genera also provide food products and medicines [4]. The commercial value for orchid flowers is very high, due to which they are in high demand all over the world. The commercial demand for orchid plants is increasing day by day; so, the improvements in orchid cultivation and their conservation can effectively develop the economy of our country.

In-vitro ANTHER, MICROSPORE OVARY AND OVULE CULTURE
Growing orchids naturally is a time-consuming and slow process [6]. Orchids have a long vegetative phase before they flower [7]. The breeding cycle of orchidaceae is also very slow and unpredictable [8]. To reduce or minimize this time period, the technique of in vitro haploid production is used. Haploids are useful in the shortening of the breeding cycle in orchids [9]. This technique is really helpful for the development of various species of orchids. Haploids are widely used in many agricultural and horticultural fields [10]. By using in vitro technique, we can develop haploid as well as diploid plants. In this paper, the production of haploid plants is being discussed.
Haploid plants are those plants that arise from gametes or gamete-like cells [11]. They do not undergo fertilization and can still generate a viable individual plant [11]. Haploid plants contain cells that have half the number of chromosomes. Haploid plants are sterile as they contain only one set of chromosomes number [12]. Haploids have only one chromosome set, that is formed after the meiosis in male or female gametes [11]. The plant is referred to as the maternal or paternal, based upon the set of chromosomes contributed by paternal or maternal side. The haploid plant is also known as monoploid because it contains only one set of chromosomes [12]. There are many advantages of haploid plants such as fixation of homozygosity, shorten breeding cycle, and high selection efficiency [13]. Haploid plants are used in the basic plant research fields such as cytogenetics, molecular genetics, crop evolution, plant biotechnology, and traditional plant breeding [14]. Haploid plants are an effective tool for induced mutagenic and genetic transformation studies [15]. In vitro techniques are an efficient way to produce haploids than other methods such as interspecific hybridization or treatment with plant growth regulators, temperature, or irradiation for haploid production [16]. In vitro methods of haploid production include-Androgenesis which is the production of haploids by the anther or pollen culture. In androgenesis, the haploid is generated from the male gamete [6]. Androgenesis is of two types-Direct androgenesis and indirect androgenesis including parent plant selection, selection of flower bud, microspore development stage determination, surface sterilization of young flower buds, isolation of anther/microspore from buds, inoculation of anther on an appropriate medium and regeneration and recovery of the plant [6]. Gynogenesis is the production of haploids by the ovary or ovule culture [6]. In gynogenesis, the female gametes are used for the production of the haploid plant. The common steps for gynogenesis include selection of the healthy parent plant, checking of ovary/ovule developmental stage by histology, selection of appropriate flower bud and surface sterilization, inoculation of ovary/ovule in a suitable medium, incubation of ovary/ovule culture, regeneration of callus/embryo and haploid plant recovery [6].
The in vitro culture technique was first given by Haberlandt in 1902. In vitro conditions in haploid production effectively reduce the juvenile period [817,18]. This technique is also called tissue culture, organ culture, and cell culture [2]. Good quality and disease-free orchid plants can be obtained by using this technique [2].
The desired plant can be selected from any forest or garden. The collected plant is then replanted in a pot filled with soil [7]. This plant is kept under in vitro conditions. The in vitro conditions provide the environment that promotes the growth of the cultured plant [7]. In vitro conditions comprises of a proper physical and chemical environment [16].
The developmental stage of microspores is crucial. Androgenesis is the production of haploid microspore and their in vitro culture. This is only possible with the immature anther containing immature pollen [16]. Juvenility has important role to play in regeneration during androgenesis [19]. Pre-treatment of anthers are the most efficient approach to the anther culture that involve a change in temperature [16]. Cold pretreatments kill non-viable microspores and anthers [16]. It blocks the microspores in the first mitosis. Thus, change the development from gametophytic phase to sporophytic phase and these pre-treatments maintain the cellular environment which are conducive for embryogenic development [16].
Culture media is a complex solution that provides suitable conditions for cell growth. Several culture media are present. Different media are suitable for different plant species such as Murashige and Skoog (MS) medium [16], Knudson (KC) medium [7], Vacin and Went medium [7], Linsmaier and Skoog (LS) medium [20] and Nitsch and Nitsch (NN) medium [16]. Among all of these media, MS medium is the most commonly used medium. The salt composition of any medium does not affect the frequency of initial division, it affects the planting efficiency a little, but it enhances embryogenesis and regeneration process [16]. The sugar content in media also shows several effects. A high concentration of sugar is crucial for inducing androgenesis [16]. The proper growth of a plant is also affected by the growth regulators, carbon source, and growth additives. The growth regulators play an essential role in deciding the regeneration pathway to be followed by explant in nutrient regime [21].
The success of in vitro production depends upon the right culture conditions such as temperature and light. The ideal conditions for in vitro haploid production are different for different species [16]. Anthers are kept under dark conditions with a temperature of 20-30°C [16]. The optimum temperature for the field grown material is usually 2°C which is higher than the greenhouse-grown material. In vitro microspore culture is all about culturing immature pollen grains that are obtained by the donor plant [22]. The methodology of microspore culture is similar to that of the anther culture [22]. This method is slower than the anther culture method for development, but the production of the embryo is higher in this method [16].
The method of in vitro haploid production has now been adopted in various plant reproducing programs world over. The methodology has emerged as an efficient technique for prompt harvest improvement for gene transfer, part of chromosome, and whole chromosomes [12,14,23,24].
Being economically stable technique, it is effectively utilised in biodiversity conservation [2]. Thus, In vitro haploid production technique is a very productive and advisable way for haploid production [6]. Haploid plants have several applications in the fields of basic research, classical plant genetics, cytogenetics, genetic transformation research, conventional plant breeding studies, genome mapping, and many more [12].
Significance: In vitro haploid production is broadly used to produce new varieties and cultivars. The technique is also employed to conduct genetic analysis of several important agricultural crop.

CONCLUSION
Orchids are the most beautiful and commercially very valuable flowers. They are very expensive. The improvement in orchid cultivation can effectively develop the economy of our country. The natural breeding cycle of orchids is very long and time-consuming. It takes several years for orchids to blossom. In vitro conditions can effectively shorten the time required of breeding cycle. In vitro haploid production can also be used to develop viable seedlings of rare, endangered, and commercially valuable orchids.
In vitro haploid production raises good-quality, orchid plant free of any kind of disease. Success of this technique is based upon identifying right stage of the capsule at harvesting time, the growth adjuvants, and suitable nutrient regime. In vitro haploid production is very useful in developing homozygous pure lines, such pure lines are utilised in various plant breeding programs, and in the research activities pertaining to plant genetics and breeding.

CONSENT
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ETHICAL APPROVAL
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