Biotechnology Laboratory for Conservation

Work Report

October-November 2015

Prepared by:

Sanjib Chattopadhyay, Senior Resarch Assistant

Debarati Chakravarty, Research Assistant

and Priyabrata Roy, Research Assistant

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Background  

There  is  consensus  among  rice  biologists  that  the  gene  for  aroma  of  Basmati  rice  likely  derived  from  an  ancient  Chinese  variety  of  Japonica  group.  A  prominent  study  (Kovach  et  al.  2009)  suggested  that  the  gene  BADH2.1,  responsible  for  Basmati  aroma,  was  derived  from  Japonica  rice.  However,  a  recent  study  by  Dr.  Avik  Ray  and  coworkers  indicated  that  the  aroma  of  small  grain  Indica  rice  varieties  probably  had  an  independent  origin  (Ray  et  al.  2013).  

 

 

Our  Analysis  

Screening  of  84  aromatic  and  non-­‐aromatic  rice  samples  for  checking  presence/  absence  of  badh2.1.  

 

Our  Achievements  

1. 22  aromatic  varieties  without  badh2.1  –  a  finding  that  challenges  the  standard  hypothesis.  

2. Presence  of  badh2.1  in  three  non-­‐aromatic  samples  (Basmati  nagini,  Cheri  gadi,  Mohanmala)  –  

further  challenging  the  hypothesis  that  badh2.1  is  responsible  for  aroma  in  rice.  

3. Submission  of  84  DNA  sequences  to  National  Centre  for  Biotechnology  (NCBI)  Genbank,  USA,  a  

public  repository,  thereby  securing  protection  of  the  sequences  from  commercial  appropriation.    

4. Procured  gene  sequence  data  concealed  from  Genbank  from  Dr.  Kovach  and  Dr.  Susan  McCouch  

of  Cornell  University  and  Monsanto  Co.,  through  the  help  of  PNAS  journal  editor,  paving  the  way  

for  further  advancement  of  research  in  origin  of  aroma  in  Indica  rice  varieties.  

5. Manuscript  being  prepared  for  publication.  

 

 

 

 

 

 

Rice  Grain  Aroma   2-­‐AP   Gene  badh2.1,  on  chromosome  8  

     Proposed  to  have  been  acquired  in  Indica  from  Japonica  (Kovach  et  al.  2009)  

1. Origin and Evolution of Aroma in Indian Rice

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References  cited:  

1. MJ Kovach, MN Calingacion, MA Fitzgerald,SR McCouch. 2009. The origin and evolution of fragrance in rice (Oryza sativa L.). Proceedings of the National Academy of Sciences of the United States of America 106:14444–14449.

2. Avik Ray, Debal Deb, Rajasri Ray and Balaji Chattopadhayay. 2013.  Phenotypic characters of rice landraces reveal independent lineages of short-grain aromatic indica rice. AoB Plants 5: plt032 (doi: 10.1093/aobpla/ plt032).

 

 

 

 

 

 

 

 

 

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2. Rice Seed Germination Enhancement In the course of collection of heirloom rice seeds, we often receive seeds that were stored for multiple

years. These seeds are virtually unviable, because the embryo dormancy cannot be broken using

conventional germination procedures. Our problem is to germinate at least a small proportion of such

seeds after longer than 2 years storage.

Constraints

1. Low germination rate after storage for 2-3 yrs.

2. Published methods claim different results

Our Experiments

     We checked 5 techniques, each with 2 variations, to germinate both wild and cultivated rice.

Results

A simple method - soaking in warm water (50 C) for 15 min significantly enhances germination probability. We

find this method to be more effective than reported methods, including treatments with hot air, hormones, and

acids. This method will be useful in germinating old seeds of rare rice varieties.

             

                                                                                                                   Rice  seeds                                                                                                    Rice  seedlings  

                                                                                                                                                                                                     

 

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3. Rescue of Critically Endangered Vitex glabrata by Tissue Culture

 

The  Plant:  Bhadu  (Vitex  glabrata)  is  a  rare  plant,  of  no  known  economic  value.  A  single  mature  tree  is  reported  from  a  fragmented  sacred  grove  of  Bankura  District  (Deb  2008;  2014).    

Status  in  Nature:    Critically  Endangered.    

Constraints:    The  seeds  do  not  germinate  in  natural  conditions  (attempted  for  15  years  by  Dr.  Debal  Deb)  

Probable  reason  of  germination  failure:  The  absence  of  the  animal  necessary  to  propagate  the  seeds.    

 

Our  Achievement    

We  succeeded  to  rescue  embryos  from  seeds,  which  we  subsequently  germinated  in  test  tube.    

 

Result  

 The  germinated  seedlings  transferred  to  soiled  pot  in  Laboratory  condition.  We  are  preparing  to  transfer  the  seedlings  to  natural  soil  condition  within  a  month.  

 

Task  Ahead  

The  saplings  will  soon  be  grown  out  in  soil.  Once  the  seedlings  are  established  in  natural  environment,  we  shall  distribute  the  saplings  to  grow  into  trees  on  Basudha  farms  in  Odisha  and  West  Bengal.  Subsequenlty,  other  saplings  will  be  germinated  and  distributed  among  interested  conservators  in  rural  and  urban  areas  and  botanic/horticultural  gardens..  

 

References  cited:  

Deb, Debal 2008. Sacred ecosystems of West Bengal, pp. 117-126. In: A K Ghosh (ed.), Status of Environment in West

Bengal: A Citizens’ Report. ENDEV Society for Environment and Development. Kolkata.

Deb, Debal 2014. The Value of Forest: an ecological economic examination of forest people’s perspective (Chapter 7), pp. 123-159, in: Trevor Fenning (ed), Challenges and Opportunities for the World’s Forests in the 21st Century. Springer: Heidelberg/New York.  

 

           

Growing  saplings  on  culture  medium  in  test  tubes  (left),  in  treated  soil  (right).  

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4. Nutraceutical Analyses of Rice

Aim:  To  explore  the  nutritional  value  of  the  traditional  rice  varieties.  

Ongoing  research  work    

Primarily,  we  are  in  the  process  of  analysing  120  rice  varieties  to  examine    

(a) total  protein  content  

(b) vitamin  B  complex  content  

(c) Fatty  acids  (including  omega  3  fatty  acid)  content  of  each  variety.    

Dr.  Mousumi  Poddar  Sarkar  of  Calcutta  University  is  collaborating  in  (b)  and  (c)  of  this  project.  atty  acids  and  

vitamin  contents  are  being  analysed  in  her  laboratory,  while  the  total  protein  contents  are  being  examined  in  

our  laboratory.    

Part  of  the  results  of  this  collaborative  study  of  fatty  acid  contents  of  60  varieties  has  already  been  submited  

for  publication  in  the  peer-­‐reviewed  journal  Current  Science.  

 

Task  ahead  

• Publish  a  detailed  research  report  on  comprehensive  nutritional  contents  of  1000  traditional  rice  varieties.  

• Establish  therapeutic  value  of  different  traditional  rice  varieties.    

 

Acid  Hydrolysis  for  vitamin  analysis.  

 

Publication  arising:  

Ray, Sandipan, Debal Deb, S. Talai Mukherjee, S. Tripathi and Mousumi Poddar Sarkar 2015. Lipid based nutraceutical value of indigenous rice (Oryza sativa ssp. indica L.) landraces. Current Science (in press).  

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5. Metal Profiling of Folk Rice Varieties

Certain heavy metals are important for nutrition in food. Moderate levels of iron, copper, zinc and molybdenum in food are desirable for a healthy diet. Until our study, comprehensive screening of folk rice varieties was lacking in scientific literature.

Our Acheievent

In collaboration with Prof. T. Pradeep of IIT-Madras, we screened 130 folk rice varieties for 12 heavy metals. We discovered more than 67 varieties that contain > 20 mg iron in every kg of rice grain, and 98 varieties with >20 mg of zinc per kg of rice grain.

More astonishingly, we detected a variety, Garib-sal, which contains substantially high (15 mg/kg) amount of silver from the farm soil. The findings are published in Deb et al. (2015).

Implications

1. Several of the iron-containing rice (e.g. Kelas, Bhut moori, Bhuri sulah) are used in tribal medicine for curing anemia in pregnant and lactating mothers. A few of these varieties (Dudhé bolta, Parmai-sal) are also used as diet for convalescing patients (Deb 2014). Garib-sal was used for treating gastro-enteric infection. The silver in this rice is likely to eradicate gut pathogens.

2. Research institutions are keen to develop genetically engineered iron-fortified (GEIF) rice, with an aim to eradicate iron deficiency in South Asian populations. The International Rice Research Institute at Los Baños, the Philippines has released IR68144-2B-2-2-3, which contains 9.8 mg of iron per kg. Our study indicates that 67 (out of 130 varieties examined) folk varieties contains greater than 20 mg of iron per kg, with at least 4 varieties containing >100 mg of iron/kg. Thus, it is pointless to invest in developing any GEIF rice, as long as so many high iron-containing rice varieties exist on farm fields.

Task Ahead

We plan to screen 1000 folk rice varieties grown and conserved in Basudha farm. Work is afoot.

Number  and  Proportion  of  Rice  Landraces  with  High  Levels  of  Metal  Concentration  in  Grains.

Publication:

Deb, Debal, S. Sengupta and T. Pradeep 2015. A profile of heavy metals in rice (Oryza sativa ssp. indica) landraces. Current Science 109: 407-409.

0   20   40   60   80   100  

Mn (>40 mg/kg)

Fe (>20 mg/kg)

Cu (>20 mg/kg)

Zn (>20 mg/kg)

Ni (>10 mg/kg)

Cr (>10 mg/kg)

Ag (>10 mg/kg)

31

67

12

98

1

7

1

% of Total No. No. Landraces

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6. Lignin Estimation of Wood of 50 Tree Species from Eastern Indian Forests

Lignin  is  an  organic  polymer,  found  in  bark  and  wood.  This  polymer  imparts  strength  and  durability  to  

the  stem  and  branches  of  trees  and  shrubs,  and  plays  an  important  role  in  natural  carbon  cycle  and  

become  humus  after  decomposition.    

 

In  sustainable  agriculture,  biochar  application  is  highly  recommended.  Biochar  quality  is  directly  

related  to  lignin  content  of  woody  biomass.  Dr.  Debal  Deb’s  research  (2015:  in  press)  indicates  that  

woody  shrubs  and  brushwood  with  considerable  amount  of  lignin  are  cheaper  and  easier  to  harvest  

for  local  biochar  production  than  stems  of  mature  trees.  This  would  obviate  felling  of  trees  and  

deforestation.  An  examination  of  ligin  content  of  wood  of  different  trees  and  shrubs  would  facilitate  

choice  of  species  for  biochar  production.    

 

Our  Work  

There  exists  no  published  data  of  lignin  contents  of  native  trees  and  shrubs  from  eastern  Indian  

forests.  We  are  in  the  process  of  estimating  the  lignin  content  of  more  than  50  native  trees,  alongside  

that  of  invasive  exotic  shrubs  (Lantana  camara  and  Eupatorium  odoaratissimum).  We  are  also  making  

a  comparison  between  species  from  Bankura's  dry  deciduous  forest  and  Orissa's  moist  deciduous  

forest.  Estimation  of  lignin  contents  of  11  species  has  already  been  accomplished.  

 

Task  ahead    

• To  publish  the  first  data  base  of  lignin  content  of  forest  flora  of  eastern  India.  

 

                     

 Lignin  estimation  after  acid  digestion  (Left);  Species  samples  for  analysis  (right).  

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7. Molecular Barcoding of a Rare Tree Likely to Be a New Species

The  Plant:  A  rare  tree,  locally  known  as  Sitapatra  has  a  distinctive  leaf  characterisic:    scratches  on  the  leaf  surface  leaves  permanent  black  marks.  (The  fluid  that  oozes  after  breaking  of  the  leaf  surface  becomes  dark  upon  oxidation.)  Sitapatra  is  a  rare  species,  with  only  23  mature  trees  occurring  in  18  sites  of  West  Bengal  (Deb  2014).  Botanists  have  so  far  failed  to  identify  this  tree  from  specimens  and  photographs  of  leaves,  flowers  and  fruits  of  the  tree.  It  appears  that  the  tree  may  belong  to  genus  Cordia,  but  Dr.  Debal  and  Dr.  Bo  Li,  a  taxonomist  from  China,  suspect  it  is  an  Ehretia  species.    

Dr.  Deb  has  documented  its  morphological  characteristics,  which  appear  to  differ  from  all  descriptions  of  known  species  of  both  Ehretia  and  Cordia  (in  Boraginaceae  family)  In  order  to  confirm  its  affinity  to  the  two  genera,  and  also  to  establish  whether  this  is  a  new  species,  we  have  conducted  molecular  taxonomic  study,  using  5  molecular  primers.  

Conservation  Status:  Rare  in  distribution  around  the  surveyed  area  (Deb  2014)  

     

From  Left  to  Right:  Sitapatra  Flower;  Fruit;  Leaf  and  Inflorescence  

 

Taxonomic  identification:  The  plants  shows  ambiguous  morphological  characters  similar  to  the  known  species  of  Cordia  and  Ehretia.  Proper  identification  of  the  plant  is  still  not  established  by  any    authentic  institution  and/or  literature  survey.  

Our  effort  

• We  have  employed  Molecular  Barcoding  –  a  cutting  edge  technique  for  taxonomic  identification  of  organisms.  

• The  primary  work  on  molecular  barcoding  (PCR  amplification  of  candidate  genes  and  sequencing  of  the  amplified  genes)  completed.  

Present  Status    

Comparing  the  sequenced  genes  as  a  molecular  signature  to  be  analysed  within  next  month.  

Task  ahead    

Making  a  consensus  among  Boraginaceae  experts,  and  publishing  the  exact  taxonomic  identity  of  the  tree  as  either  a  new  species  or  a  variant  of  a  known  species.    

 

Reference  cited:    

Deb, Debal 2014. The Value of Forest: an ecological economic examination of forest people’s perspective (Chapter 7), pp. 123-159, in: Trevor Fenning (ed), Challenges and Opportunities for the World’s Forests in the 21st Century. Springer: Heidelberg/New York.