Coconut Leaf Tissue Culture
Differentiation of Embryoids and Development of
Preface - The elaborate work on Coconut [ Cocos Nucifera] Cloning was done nearly two decades back during 1981 to 87, along with other plants such as cashew and few woody trees. My interest also extended to certain innovative concepts and ideas. In spite of very decisive progress towards cloning coconut, I had to retreat from the scientific world. On the eve of submission of my doctoral thesis, after nearly seven years of intensive research, I quit competitive world of research to stay with my consciousness than submitting it in exchange for a doctoral degree and my future in academic research. The past one and half decades, my attention was directed to the fundamental questions of science as freelance researcher and as a small agriculurist.
Today, I am choosing to share my experience in coconut with a caution. Information in biological systems is qualitatively and quantitatively dynamic. They are not stable. Clonal propagation through somatic embryogenesis will not give the desired results but the work has other potentials for application.
This article is just an attempt to share in brief, the tendencies I observed while I was dealing with the system. My interest now stands much beyond tissue culture and biotechnology. It rests in the fundamental design and principle of nature that would lead to reorganize the foundation of health, agriculture and social thinking and building of the modern world such that it becomes compatible with nature and brings forth order and peace to nature and humanity.
[First stage of work]
Method and Approach: The method and approach fallowed was rather innovative. Normal approach followed by the researchers in this field is to check methodically the different combination of hormones on a selected media. It is assumed that ratio favoring Auxins produces root, a ratio favoring Cytokines produce shoot growth and right combination of them also can lead to somatic embryogenesis. Success is related to large number of trials and chance. No body knows even vaguely what drives the dedifferentiation and differentiation of information. The concentration of Auxins/Cytokines used normally ranges 0.1mg to 5mg/liter.
The searching process adopted by scientist ingeneral appeared really a hopeless one when it came to a plant like coconut. A year’s day and nights work and hundreds of different combination charted out for me by my superior, produced no effect on the leaf material transplanted to the media. I was about to give it up and quit my research.
Luckily, I developed a strange communication with Nature and it started opening the paths for me. She called upon me to use my senses something that science never uses.
The methodology I used was to observe the concerned plant in nature and note down various different Physico–chemical factors affecting it. This involved constant interaction with farmers, specially experienced ones. My back ground as a farmer also helped it. The information obtained was pooled and was intropsected on and used as a base to put up random experiments. Nature helped me design a new experimental design. The goal here was kept centered.
New Experimental Design
Comparison between Normal and the New Design
The normal approach begins at a point [small concentration of various hormones] and extends to scanning a infinite combinations
Note - this canbe used universally to quicken the the research work on any species and can save lots precious resources.
The new approach was random. Here the goal was kept centered. It is principally built on the simple observation that the living system is stressed in nature to dedifferentiate from time to time in order to survive. The principal stress can be deduced to heat and heat is a spatial winding and unwinding force. [This aspect will be discussed greatly in dynamic information and its survival in nature, yet to be loaded to the site]
The experimental design goes as below
Effect of a chosen hormone or the component of the medium is determined by a series of nine experiments that are grouped into three - minimum, maximum and optimum.
For example to investigate the effect of NAA - I would keep 3 experiments.
1] Without the hormone [Control]
2] With 1mg/l of NAA
3] With 10mg/l of NAA
4] With 100 mg/l of NAA
There is a minimum, optimum and maximum. The observation would guide me to the next step. If the system responded below 10mg/l, the next step of experiment would be to determine three more levels within it.
In the second step I would keep
1] With 2.5mg/l of NAA
2] With 5 mg/l of NAA
3] With 7.5 mg/ of NAA
Three more experiments are laid to fine tune the concentration. If the response in the first step is above 10 mg/l, then above process is repeated to find the concentration and fine-tune it
The process is repeated for the opposite hormone [Cytokines] and different combinations of them. This rapidly decreases the number of experiments and increases chances of obtaining favorable tendencies to work with.
For checking the influence of Macro and micro nutrients, same approach was adopted.
The advantage of this design was that I could quickly get into a path that would lead me to the goal. The observation and communication I developed with nature and my back ground as an agriculturist helped me choose right path instinctively. For example
· The reaction of plant to excess application of potash and nitrogen fertilizers helped me fix and vary the nitrogen content and potash content of the medium.
· The observation that partially dried nut germinated better in the nursery, helped adopt certain practices at the lab level to increase the germination rate of coconut embryos in Vitro.
Material and Method
Leaf Tissues isolated from 1-2 year old seedlings. About 6 to 8 cm length of the innermost 2 to 5 leaf was used for the experiment. They were cut into 1 cm pieces with 3 or 4 folds of leaf and transferred to the media. The media used in the beginning stages was Eeuwen's media.
In the random experimentation process those experiment where the NAA concentrations increased to abnormally high levels 60-120mg/l, pieces of tissues showed surprising capacity to survive and grow in the medium. It is rare in tissue culture experiments to use auxins concentration above 5mg/l
Increasing the sugar concentration also showed positive signs. In a basal medium with 90gm/l of sucrose and 90mg/l of NAA, the leaf pieces started producing globular structures at the basal cut end of the leaf pieces. Each vein of the leaf ended up producing one globular structure, which readily formed into roots. See fig 1-6. These structures were isolated in the early stages and were grown in medium containing different Cytokines. However, they tended to produce good roots and all attempts to manipulate these structures to produce shoots by varying the Auxins and Cytokinins ratio failed.