Coconut Leaf Tissue Culture

 
Cloning Coconut
 
Coconut Leaf Tissue Culture
 

 

Differentiation of Embryoids and Development of
 
Cloned Plants

  

 

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  
 
New Approach
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.

 

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 1-6.  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.

     
Fig -1                                                 Fig -2                                         Fig-3
 
 
 
    
Fig-4                                                       Fig-5                                         Fig-6
 
Fig 1 to 6 shows coconut leaf giving rise to embryoids in high NAA and Sugar contianing media and attemps to isolate and grow  them resultinig root formation.  
 
 

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.  7 to 9

 

      
Fig-7                                           Fig-8                                              Fig-9
 
Fig -7 to 9 shows predominant cellular differrntiation leadin to formation roots in high NAA and sugar contianing medium.
 

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 10 and 11 [Morphology] and Fig 12-19 [Cytology]
 
     
Fig -10                                                              Fig-11
 
Fig –10 and 11 : Different stages of embryo growth in vitro and its comparison with endosperm
 
    
Fig-12                        Fig-13                        Fig-14                     Fig-15 
 
 
 
     
Fig-16                                                                Fig-17
 
 
Fig-18                                                        Fig-19
 

Fig 12 to 19, how embryo in-vivo differentiates and begins to germinate. Note there is very little differentiation of vascular tissues in the early stages 

 

 

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 -12, 13, 14, 15

From the limited observation of these section studies I could figure out a possible physical and 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- 16 and 17

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 - 18. 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  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 by supressing the diferntiation of cells into conducting tissues. 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.

 

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. This necessitated taking a differnt path. 

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 to retain the stress but change its influence on the system. 

Embryoids formed now showed clear change in their morphological and histological appearance. See figure- 20-28

 

   
Fig-20                                          Fig-21                                         Fig-22
 
 
   
Fig-23                                          Fig-24                                           Fig-25
 
   
Fig-26                                           Fig-27                                      Fig-28
 
 
Fig 20 to 28 shows differnt types embryoids obtained from coconut leaf in-vitro in various combination of sugars. They showed morphologically and histologically resemblence with embryos growing and differentiating in-vivo    
 
 
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-26,{morphology} 38, 39, 40, 41 Histology], and also from cells beneath it [Fig-25]. In some cases these embryoids like structures in turn started produce more embryoids like structure that appeared as globular callus. See figure -23, 24.  All of them were subjected to section studies. See figure below. 29-37
 
  
Fig-29                                              Fig-30                                      Fig-31
   
         
Fig-32                                           Fig-33                                        Fig-34 
 
  
Fig-35                                           Fig-36                                                Fig-37 
 
 
Fig 29 to 37 shows histological studies of In vitro embryoids obtained from leaf that closley resemble in vivo development fig 12 to 14. Note marked control of differntiation of vasular tissues and tendencey to form shoot primordia with lef life structures fig -34 and 37
 
 
 
 
Fig -38                                                            Fig-39
 
 
 
Fig-40                                               Fig-41 
 
Fig 38-41 shows the tendency of epidermal cells of leaf to differentiate to form embryoid. Note  fig -26 where such embryoids showin tendency form root and shoot.
 
 
 Fig-42                                                                          Fig-43
 
 
         
        Fig-44                                                                    Fig-45  
 
Fig -42 to 44 shows a collection of embryoids that detech and fall into the meida and one of them germinating. Fig 44 show a rare case of multliple shoots
 
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.

 
Though only two batches of my experiment the embryoids gave rise to well formed shoots and resulted in a plant [the plant was lost through infection] the result was encouraging. 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 central zone of my goal. All it needed was some concentrated effort and fine-tuning. I was extremely confident of developing a clonal technology for coconut.  

 

 Third Stage that got Truncated

The third stage was planned to fine tune the results to get consistent and improved results. The approach was to study the Physico-chemical properties of the milieu in which coconut embryos grows In-Vivo in depth and mimic it. It is important to note that the fertilized egg takes 12 months to mature in varying physico-chemical consitions.

 

The work was in its initial stages when unfortunately I was caught in a tempest that virtually shook me and I decided to leave the competitive world. Two factors prompted me to take this path

  • I became victim of greed and "self" of my superior, whom I believed as a son believes his father.   In 1985, the work hit the head lines of news media, where I was reduced to a lab assistant to my superior whose actual intellectual participation was absolutely zero. The work was over exaggerated and was virtually misleading. I resisted this approach by my superior. Eventually on the eve of submission of my Doctoral Thesis when I was asked to bow my head to get that vital signature to my thesis. I chose to quit and stay with my consciousness than bowing to a person who wears white clothes, speaks of truth and takes prominent position in society but carries dark intentions.  
  • I took to research from consciousness hoping to contribute something to humanity. But during the process of my research I realized that the foundation of biotechnology and our knowledge of life are wanting. The foundation of modern biotechnolgy exists on information that is invariable. My observations in the lab and my communication with nature was speaking other wise and disturbing my consciousness and challenging my intelligence to uncover her truth. A small stint with a multinational company exposed the way our research thrives. At this point, became more inclined to take academic research. But when the passport to academic research demanded sacrificing the very consciousness and the principle on which I stood, I chose to quit, but pursue my enquiry into nature independently. 

 

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.  But this physico-chemical factor contributing to dedifferentiation 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 stress 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 seem to have broken this condition of directed flow in the leaf tissues and suppressed the cell maturation and differentiation of conducting tissues. This led to embryoids like structures forming from the epidermal cells. It appears that the osmotic conditions of the media play 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 differentiates information in space and time. However form time o time it dedifferentiates its information and initializes it against time. This 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 of they system against time. Herr we dedifferentiate the system to the near zero state but over step the union of two different systems and quantum collapse and reorganization in space and time. Thus we interfere into natural process of the system that helps it conquer time and death.

 

The plants animals seem to work in relation to minute changes in nature and its time cycles. The information in them is not paired as we think, but are dynamic units containing minimum of 4 pairs of information’s. 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.

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