1. Teknik & Aplikasi Kuljar
Dalam Produksi & Kualitas Tanaman
“… the art and science of multiplying plants in vitro.”
Dr. Sukendah, MSc

2. Pembentukan Benih Sintetik
Benih sintetik (Synthetic/Artificial Seed) adalah produksi benih dari embrio somatik yang dibungkus dalam mantel tiruan
Artificial
seed coat
Somatic embryo
Artificial endosperm

3. Teknik Pembuatan Benih Sintetik
MS medium + phytohormone + carbon sources.
Sodium alginate was added to the medium and mixed well to make a homogenous solution.
The explants were taken and dipped into the alginate solution.
Explants dipped in alginate solution were taken up by a forcep and placed to the beaker of CaCl2
Each explant then became a hard ball encoded by alginate.

4. Teknik Pembuatan Benih Sintetik
Bahan Benih:
Embrio Somatik
Seed
3% Sodium alginate + MS free calcium salt solution
Sodium alginate
Calcium cloride
Pembentukan seedcoat
Seed Coat
75 mM CaCl2.2H2O
Artificial Seed

5. Haploid Plant Production
Embryo rescue of interspecific crosses
Creation of alloploids (e.g. triticale)
Bulbosum method
Anther culture/Microspore culture
Culturing of Anthers or Pollen grains (microspores)
Derive a mature plant from a single microspore
Ovule culture
Culturing of unfertilized ovules (macrospores)
Sometimes “trick” ovule into thinking it has been fertilized

6. Anther/Microspore Culture

7. Anther/Microspore Culture Factors
Genotype
As with all tissue culture techniques
Growth of mother plant
Usually requires optimum growing conditions
Correct stage of pollen development
Need to be able to switch pollen development from gametogenesis to embryogenesis
Pretreatment of anthers
Cold or heat have both been effective
Culture media
Additives, Agar vs. ‘Floating’

8. What do you do with the haploid?
Weak, sterile plant
Usually want to double the chromosomes, creating a dihaploid plant with normal growth & fertility
Chromosomes can be doubled by
Colchicine treatment
Spontaneous doubling
Tends to occur in all haploids at varying levels
Many systems rely on it, using visual observation to detect spontaneous dihaploids
Can be confirmed using flow cytometry

9. Created by degrading the cell wall using enzymes
Protoplast
Created by degrading the cell wall using enzymes

10. Protoplast fusion
Protoplasts are made from two species that you want to cross
The membranes are made to fuse
osmotic shock, electrical current, virus
Regenerate the hybrid fusion product
Contain genome from both organisms
Very, very difficult

12. Thank You