The effect of different concentrations of some plant growth regulators on the micropropagation of orchids (Orchids catasetum)

Dissertation for M.Sc.

Abstract

Orchids are among the most diverse family of flowering plants. This family has more than 800 genera and 25,000 species. Orchids come in a wide variety of sizes, shapes and colors. The lifespan of orchids after harvesting is also long. The large-scale increase of orchids using plant tissue culture techniques contributes to the position of this plant as one of the top ten flowers in the world. In this research, a guideline for the propagation of orchids (Orchis catasetum) in glass is provided. Pseudoprotocorm bodies (PLBs) were cultured as explants on Morashig and Skoog (MS) medium enriched with different concentrations of benzyladenine (BA), indole butyric acid (IBA) and naphthalene acetic acid (NAA). A combination of 0.5 mg/L BA together with 0.5 mg/L NAA was suitable for inducing the maximum regeneration of PLBs (20.40 per seedling). Also, in this environment, the highest number of roots (7.16 per seedling) and leaves (10.10 per seedling), as well as the highest seedling length (114.20 mm per seedling) and root (193.40 mm per seedling) were observed.

Key words: orchid, auxins, pseudoprotocorm bodies, cytokinins increase within the tubes

Foreword

One ??of the most important techniques of tissue culture, which has been widely spread due to its significant benefits, is the propagation of plants through vegetative embryogenesis, and this method is very useful for the production and regeneration of valuable plants on a large scale and at a very low cost (Mashayakhi, 2016). More than a hundred years ago, in 1902, the German physiologist Haberlent claimed that the formation of embryos from vegetative cells is possible, and his research led to the establishment of in vitro culture methods (Halperin, 1995; Kielenbach, 1984). This theory was not proven for 55 years until it was reported for the first time by Reinert in Germany and Steward and his colleagues at Cornell University in America in 1958, without these two researchers knowing about each other's research, about the occurrence of vegetative embryo formation through callus culture and also carrot root (Ducus carota) cell suspension culture (Reinert, 1958; Steward, 1958).

1-1-Botany and ecological distribution

The orchid with the scientific name orchids catasetum belongs to the orchidaceae family. According to many scientists, orchid flowers are among the most evolved plants. The fox family is considered one of the richest plant groups in terms of diversity with more than 600 genera and 2000 species in the world (Mohammadian, 1376).

Orchid family (Orchidaceae) from the Gynandrales order. The plants of this family are monocotyledonous, herbaceous and perennial, which can be seen as parasites, saprophytes and soil plants (Mohammadi, 1376).

The ecological distribution of this plant goes back to Southeast Asian regions such as China, Taiwan, Hong Kong, Thailand, Philippines, Malaysia, and Singapore.

Orchids are the best-selling and most attractive plants for homes, apartments, restaurants, hotels, and other public centers in the whole world today, especially in Europe, America, and Asia. This popularity has been achieved for two reasons. The flowering season and flowering time of orchids is different, usually it starts from December and they have flowers for several months. After flowering, some rest and regeneration is needed to flower again. There are many different plant species in the orchid family. About 12 to 15 different varieties of orchids are cultivated in our country. (Mohammadian et al., 1376)

1-2- Economic importance

Orchids are the best-selling and most attractive plants for houses, apartments, restaurants, hotels and other public centers in the whole world, especially in Europe, America, and Asia. This popularity has been achieved for two reasons. First, these plants are very low-maintenance and adapt to the environment with little maintenance. On the other hand, when they flower, their flowers remain beautiful and bright for months (about three to six months depending on the environment and conditions).(Khushkhovi, 2009)

1-3- Reproduction method

Orchids are propagated in two ways: vegetative and reproductive. In vegetative reproduction, the progeny are the same as the parents; But in reproductive propagation (by seed) the same progeny are rarely obtained, and the concern is mainly when wild species are considered. Therefore, if the seeds of orchid domestic varieties are used, which are usually the result of crosses and are strongly heterozygous, the progeny will be very non-uniform, and less like the original plants. Basically, cultivated orchid cultivars are propagated only through vegetative means. Preparing an orchid clone in vivo is a very slow process, and sometimes it takes 10 years to get a certain size of an orchid clone. After 1960, there was a revolution in the vegetative propagation of orchids (Mohammadi et al.). (Morrell, 1960) tried to obtain a virus-free Cymbidium leaf through meristem planting, and for this purpose, he used the terminal parts of the stem in vitro culture. The initial corm-like forms obtained from aerial parts were very similar to those previously obtained from seeds. In some cases, these primary corms divide spontaneously; But usually this operation is done by cutting the primary corms into smaller pieces (Morrell, 1965). In Cymbidium plant, within six weeks, it is possible to produce 6 to 8 primary corms from one corm. In principle, the division of these primary corms can continue indefinitely. When the work of division is finished, each primary corm can produce aerial organs, leaves and roots. In this way, Morel was able to produce thousands of plants (a meristem with a few primary leaves) from a single shoot in a year. Orchid clone production by meristem culture was the first commercial asexual propagation in this in vitro culture medium. This method was originally done for Cymbidium plant, which is used with modifications in Cattleya plant and many other orchid species.                             

When the primary corm is formed after germination of an orchid seed, it has a morphological state intermediate between an undifferentiated embryo and an aerial stem. Forms similar to primary corms are formed by vegetative propagation with meristem planting. In fact, in this way, the terminal part of the aerial part of a mature plant is rejuvenated (returning from the mature state to the young state). Since the primary corms obtained from seed germination are very similar to the primary corms obtained from meristem planting, for this reason, the term primary corm-like organs is used in orchid clones by meristem cultivation (Champagnet, 1977). The vegetative propagation of orchids by means of meristem cultivation can be divided into three stages:

1- Converting the meristem into primary corm forms

2- Propagation of primary corms by cutting them into smaller pieces

3- Their growth until the production of rooted stems (theo , 1978, 1976). 

1-4- Statement of the problem and purpose of the research

Plant tissue culture refers to the growth of plant material free from pathogenic agents in disinfected conditions and in a sterile food bed in laboratory containers such as test tubes. Plant tissue culture includes the culture of radioplasts, cells, tissues and plant organs (Bagheri et al., 2013). The biggest incentive for using plant tissue culture techniques for propagation of many species may have been the early work of Morel on orchid propagation in 1960 and the work of Morashik and Esgok in 1964 in creating a new culture medium (Moeini and Kehrizi, 1382).

Abstract:

The application of modern biotechnology for mass propagation of endangered species needs to develop a proper in vitro protocol. Here, a protocol was developed for high frequency in vitro multiplication of an endangered orchid, Orchis catasetum. Protocorms, as explants were cultured on Murashige and Skoog (MS) medium fortified with different concentrations of N6-benzyladenine (BA), ?-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) either individually or in combination. A combination of 0.5 mg l-1 BA and 0.5 mg l-1 NAA was found to be suitable for maximum protocorm-like bodies (PLBs) regeneration (20.40/plantlet). The maximum number of root (7.16/plantlet) and leaf (10.