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Cloning Coconut 

Coconut Leaf Tissue Culture

 

Differentiation of Embryoids and Development of Cloned Plants

 

By John Paily

 

 

Related Page - Coconut Embryo Culture

Related Page- Coconut Cloning an Introduction

Preface - The elaborate work on Coconut Cloning [ Cocos Nucifera cloning] was done nearly two decades back in 1981 to 87, along with other plants such as cashew and few woody trees. 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. 

Today, I am choosing to share my experience with coconut with a caution. Information system in biological system 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 it in brief, the tendencies I observed while dealing with the system. My interest now stand 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 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.  

This searching process appeared really a hopeless one when it came to a plant like coconut. A year’s day and nights work and over 1000  different combination  charted out for me produced no effect on the leaf material transplanted to the media. I was about to give 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 used as a base to put up random experiments. Nature helped me design a new experimental design. The goal here was kept centered. 

Comparison of Normal Approach and the New Approach

           

The normal approach begins at a point [small concentration of various hormones] and extends to scanning a infinite combinations   

New Approach

The new approach was random where 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 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.

Results

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 1-8.  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.  

 

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Cytological Study

Cytological study undertaken to determine the developmental pattern revealed that these structures takes an independent origin from a single cells of phloem tissues and that these structures showed a predominant tendency towards cell maturation and formation of conducting tissues.  See fig.  8A-C

Fig-8A

Fig-8B

Fig-8C

When all attempts to obtain shoots from these structures failed, I went back to nature to collect the information and study the natural process in detail. Immature embryos from tender coconuts of different stages of growth were isolated and their section studies were conducted. See Fig 9A and 9B [Morphology] and Fig 10-17 [Cytology]

 

Fig-9A

Fig-9B

Fig – 9A and 9B : different stages of embryo growth and its comparison with endosperm

 

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The observation showed that the differentiation of fertilized egg into fully mature embryo goes in a condition of constantly changing physico-chemical conditions. From fertilization to maturity it takes 12 month. The early growth occurs in a liquid medium and then it gets enclosed in semi-solid state. The earliest state that could be handled for cytological studies was taken from 6 to 7 month old tender coconut, where the endosperm was in a semi-solid state and the embryo appeared small whitish globular structure of size of a grain of sand. Cytological studies of these embryos showed no tissue maturation. At these early stages they seem to show a distinct a tripolar development with a shoot, root and haustorial end. The shoot meristem seems to develop by a process enfolding. See fig -10, 11, 12,

From the limited observation of these section studies I could figure out a possible physical geometrical process of energy unfolding and thereby development of embryo in coconut. Embryo differentiation in coconut seems to have perfect symmetrical trihedral 4dimensional process that later transform two dimensional one. This is discussed in "A Possible Geometry and Energy flow in Developing Embryo of Coconut"

Cytological studies showed that, the embryos developing in-vivo [in nature] show no tissue maturation and differentiation. A shoot meristem is the first thing to be formed. Tripolarity comes into existence very early in the embryo growth and tissue multiplication occurs from the center such that the apical shoot meristem and root meristem get embedded into the embryo with many leaf primordia.  A cross section of the immature embryo of a size little bigger than pin head [6 -7 month old] shows a distinct shoot meristem with many leaf primordia. See fig- 14 and 15

A distinct root meristem seems to develop very late in the embryo development. It becomes fully evident at the mature state. The mature embryo has well marked tripolarity. It has a structure resembling letter “T “. The vertical component represents the haustorium and the horizontal component holds the shoot and root meristem. See fig - 16. Even a mature embryo has very little tissue differentiation into phloem and xylem tissues [Conducting tissues]. The tissue differentiation occurs only when the embryo starts germinating.

From these studies it became clear that 

1] The medium and the environment in which I am trying to produce embryoids is favoring to tissue maturation, differentiation into transporting tissues and that the physico-chemical environment in which it was grown was entirely in favor of root formation. 

2] The fact that these embryoids are formed at the lower cut end of the leaf invariably means that some physical phenomenon that related to the flow and accumulation some factor within is aiding the development embryoids and its differentiation into roots.

This meant that I must reformulate the medium to suppress the tissue differentiation and break the remnant mechanical force of directed flow that exists in the leaf material in order to control the differentiation process.

 

Coconut Leaf Tissue Culture – Second stage

The hurdle for the second stage was now well defined. Work had to progress in the line of controlling the differentiation of the tissues to favor the shoot formation. The task now turned more difficult. In nature [In-Vivo] embryo of coconut is nourished and developed gradually over a period that spans up to 12 months. In this span the physico-chemical property in which the embryo matures shows changes. [Initial attempts at growing of immature embryos were not all that encouraging]. A detailed study of physico-chemical changes occurring in the tender coconuts as it matures became a necessity. However, this was beyond the capacity of the small lab in which I was working. The available information suggested that the coconut water is a rich source of inorganic ions, amino acids, organic acids, vitamins, various sugars and growth substances [ V.Raghavan 1976, “ Experimental Embryogenesis in Vascular Plants” Academic Press, London]

Knowing very well that the task in hand is rather difficult one, I choose to proceed carefully. No detailed information existed about the monthly/periodic changes in the physico-chemical properties of liquid endosperm in which the fertilized embryo differentiates to form a full grown embryo. So once again I rested on my senses to make vague assessment and from its premises began to design further random experimentation. I tasted the tender coconut water of different stages of growth and found the taste differing. The following observation became the pivot for further experimentation.  

1] The period when the embryo gives rise to shoot meristem and goes ahead to form many leaf primordia; it is enclosed in a semisolid liquid endosperm. The existence, inorganic ions, amino acids, organic acids and sugars invariably meant the osmotic state of the milieu should be at high end

2] The liquid endosperm or tender coconut water, at that stage tasted much different from mature coconut water. It was less sweet than mature coconut water and thus was concluded that it had other form of sugar in higher concentration than sucrose.

3] Since tender coconut was huge source of all amino acids, it was assumed that nitrogen content of the milieu should be high

4] Work on embryo culture has already shown that high nitrogen content reduces the germination, possibly reducing the internal cell differentiation into conducting tissues. In Contrast sucrose concentration was found to increase the root growth.

Since the main tool that led to the dedifferentiation was the increased sugar concentration and high concentration of NAA. Both these factors were essential for the dedifferentiation of information but both are known to promote cell maturation and tissue differentiation and formation of conducting tissues.

The fact that the coconut contains various forms of sugar made me to work on various types of sugar, their concentration and vary the total nitrogen content. The basic idea was retain the stress but change its influence on the system. 

Embryoids formed now showed clear change in their morphological and histological appearance. See figure- 18-26

 

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Fig-26

The tendency for cell maturation and differentiation into conducting tissues were controlled. More than sixty different combination produced embryoids like structure. Embryoids like structures began to form not only at the cut end but on the surface from epidermal cells [Fig-24,{morphology} 36, 37.38, 39 Histology], and from cells beneath it [Fig-22]. In some cases these embryoids like structures in turn started produce more embryoids like structure that appeared as globular callus. See figure -20, 21.  All of them were suubjected to section studies. See figure below. 27-35

 

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Few combinations which showed immense promise were corned. Embryoids formed in these combinations very closely resembled the one forming In-Vivo. See fig 27-31, Cytology, Fig- 40 Morphology

These embryoids get detached and fall in to the medium as independent globular structures See 40,  and resembled  morphologically and cytologically [See figure 27 and 31], the early stages of in-vivo embryos isolated from tender coconuts. 

Section studies of globular embryoids falling into the medium showed tendencies towards the formation of shoot meristem and leaf like structures see figure 30-31.  But a well-developed shoot meristem with many leaf primordia was still lacking. From among thousand of test tube, one set of experiment containing a globular mass of cells, gave rise to two shoots [See fig - 33].  In another case one particular set of experiment embryoids originating from cut end of the leaf tissues gave rise to root and shoot and a complete plant [See fig 41-42]. But the process of differentiation and germination was still not in control.

The importance of the work comes from the in depth study and approach. Beyond doubt the work was in the right direction and that I have touched the zone of my goal. All it needed was some concentrated effort and fine-tuning. I was extremely confident developing the technology.  

 

 Third Stage that got truncated

The third stage was directed at an in depth study of the Physico-chemical properties of the milieu in which coconut embryos grows In-Vivo. It is important to note that the fertilized egg takes 12 months to mature. There was a necessity to fine-tune the work. Work in this direction was already in progress, when the human greed for name power at all cost produced the tempest and I was thrown off the center. I had to retreat from the scientific world. In 1985, the work hit the head lines of news media where I was reduced to a simple technical assistant to my superior whose actual intellectual participation was absolutely zero.

My interest in cloning collapsed not only for the deep blow I received from my superior, a priest, whom I believed as a son believes the father but also for the fact that, deep within me I started to perceive that the very foundation of biotechnology is some how is wrong. The observation of living parts in thousands of test tubes and my communication with nature was clearly pointing to it.  A short stint with a multinational company where I got an exposure to the larger scientific world revealed the corruption in scientific world, especially the biological science whose fundamentals still exist undiscovered. The turbulence in me boiled to a point of no return on the eve of submission of my doctoral thesis [for which I sacrificed the most]. I was asked by my superior, to submit in writing that all the work done is his intellectual property and I was only a technical assistant to him in return to that important signature I needed to submit my doctoral thesis. I retreated than sacrificing my consciousness and dowsing the fire with in me at the feet of a man whom I discovered as less than ordinary and lacks the credentials of a good man let go a good priest or a scientist.

Sacrificing the doctoral work meant I sacrificed my academic career. A job back in multinational companies was beckoning but my heart not was willing. A welcome rest in my village before I could take a future direction turned crucial. My communication with nature become intense and it opened up answers to certain fundamental questions that were disturbing me. Slowly I began to like the freedom of enquiry and turned the whole nature into my lab. I became silent observer of nature and the multitude of experiments that are going on in it. This gave shape to the dynamic vision of biological system and eventually led me to the Unification Theory

Discussion of the Result and Observation of Coconut Cloning Effort

The fact that the embryoids like structure was obtained in the lower cut end in the initial experimentations, clearly speaks of accumulation some factor within the pieces of the tissue playing vital role in the end result.   It appeared that this physico-chemical contributed the condition for dedifferentiation, but did not facilitate orderly differentiation into full fledged embryo, instead it supported quick cell maturation and differentiation of conducting tissues. The attachment with parental tissue appeared to have some influence on the observed result.

Else where working with cashew seed I accidentally observed such situation where factors flowing and accumulating and stressing some parts of the system stressing the cells of that part of the system to dedifferentiate to form embryoids. But when the  they are still in contact with the parental system and its flow they tend to assist the parental system to survive by forming shoot and root than to making an independent system of it self. See the article "The Seed that changed My Life"

Later experiments seems to have broken this condition of directed flow in the leaf tissues leading to embryoids like structures forming from the epidermal cells and suppressing the cell maturation and differentiation of conducting tissues. It appears the osmotic conditions of the media plays a vital role. Measuring and controlling the osmotic condition of the media appears vital to control the development and differentiation. There appears a necessity to develop a method to do it and incorporate it in tissue culture experiments.  I hope such incorporation would give more stability and control over the dedifferentiation and differentiation process.

No discussion on cloning can be complete unless we understand the process of dedifferentiation and differentiation of information. A plant system for that matter all biological system is differentiated information in space and time that gets initialized from time to time through a process of dedifferentiation that involves mixing between two system a male and female or positive and negative or left and right. The whole truth of dedifferentiation and differentiation, its necessity, cause and factors determining it, is little understood. When we clone a living system we are over stepping the natural process of mixing of information and initialization by pushing the system to near state "0" and stressing it take the less preferred state of perpetuation without mixing. 

The act of dedifferentiation and differentiation is expressed in the following figure

     

A differentiated plant system is a system that exist in two dimensional in space[ a flow moving from space to earth and flow moving from earth to space], but dedifferentiation takes it to a state  of four dimensions before collapsing back in to two dimension. This means there is an initialization of time at which tissue looses two dimensional flows before it regains the direction again. The process of dedifferentiation is simply a method of stressing the flow in the system back to its "0" state where it collapses into new state changing its left and right. These aspects will be discussed in the site as the time permits in great detail. There is a need to review the very concept of cloning from the premises this knowledge of life and its fundamental process.

Note - Today I carry no hatred against my superior, in fact I look at him as a tool used by the unseen to direct my  path to Greater Secret of Nature.

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